Methods for dna-dependent targeting of a cell permeant antibody

ABSTRACT

The invention provides methods for selective targeting of live cells, which have undergone or are undergoing radiation or chemotherapy, at a site of interest with a cell-penetrating polypeptide. In one embodiment of the invention, the method comprises contacting the live cells with a cell-penetrating polypeptide comprising cell-penetrating determinants so that the cell-penetrating polypeptide binds extracellular DNA near or around the live cells so as to form a complex or association therewith such that the complex or associated polypeptide-DNA so bound bind the live cells and penetrates the live cells thereby selectively targeting live cells at a site of interest with a cell-penetrating polypeptide.

The subject application claims the priority of U.S. Ser. No. 62/117,694,filed Feb. 18, 2015, the disclosure of which, in its entirety, is herebyincorporated by reference into this application.

This invention was made with government support by the United StatesDepartment of Veterans Affairs. The government has certain rights in theinvention.

Throughout this application various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

A select group of lupus anti-DNA autoantibodies penetrate into livingcells (1), and one unusual lupus autoantibody that penetrates cellnuclei without causing any apparent harm to normal cells or tissues,3E10 (2), has been developed as a molecular delivery vehicle.Specifically, a 3E10 single chain variable fragment (scFv) with anenhancing mutation in CDR1 that increases DNA binding and efficiency ofnuclear penetration has been used to carry cargo proteins including p53,Hsp70, and other antibody fragments into cell nuclei in vitro and invivo β-6). 3E10 scFv also has activity by itself and has been shown toinhibit DNA repair, sensitize cancer cells to DNA-damaging therapy, andto be toxic to BRCA2-deficient cancer cells (7). 3E10 scFv has potentialto be used in molecular therapy approaches to diseases ranging fromcancer to ischemic conditions such as stroke, and a greaterunderstanding of the details of the mechanism by which it penetratescell nuclei is important to further delineating the scope of itstherapeutic applications.

Mutations in 3E10 that interfere with its ability to bind DNA alsorender the antibody incapable of nuclear penetration. In addition, 3E10scFv has previously been shown capable of penetrating into cell nucleiin an ENT2-dependent manner, with efficiency of nuclear uptake greatlyimpaired in ENT2-deficient cells (8). Taken together, these findingssuggest a link between cellular uptake of DNA and nuclear penetration by3E10 scFv. Interestingly, when a 3E10 scFv-Hsp70 fusion protein(Fv-Hsp70) was administered intravenously to rats three hours afterligation of middle cerebral arteries to induce stroke, Fv-Hsp70 wasfound to selectively localize to regions of ischemic brain (9).

The invention involves the discovery of the mechanism by which someanti-DNA antibodies or fragments thereof penetrate the cell for use inbetter treating disease, disorders and conditions.

SUMMARY OF THE INVENTION

The invention provides methods for selective targeting of live cells,which have undergone or are undergoing radiation or chemotherapy, at asite of interest with a cell-penetrating polypeptide. In one embodimentof the invention, the method comprises contacting the live cells with acell-penetrating polypeptide comprising cell-penetrating determinants sothat the cell-penetrating polypeptide binds extracellular DNA near oraround the live cells so as to form a complex or association therewithsuch that the complex or associated polypeptide-DNA so bound bind thelive cells and penetrates the live cells thereby selectively targetinglive cells at a site of interest with a cell-penetrating polypeptide.

Additionally, the invention provides method for selective targeting oflive cells at or near the proximity of a cellular injury with acell-penetrating polypeptide which comprises cell-penetratingdeterminants joined to, or combined with, a therapeutic agent. In oneembodiment, the method comprising administering the cell-penetratingpolypeptide at or near the proximity of the injury so that it bindsextracellular DNA from the cellular injury so as to form a complex orassociation therewith such that the complex or associatedpolypeptide-DNA-therapeutic agent so bound bind the live cells andpenetrates the live cells thereby selectively targeting live cells at asite of interest with the cell-penetrating polypeptide.

The invention further provides methods for selective targeting of livecells at or near the proximity of a cellular injury with acell-penetrating polypeptide. In an embodiment of the invention, thecell-penetrating determinants are joined to, or combined with, atherapeutic agent. The method may comprise contacting the live cellswith a composition having (a) a cell-penetrating polypeptide whichcomprises cell-penetrating determinants and (b) extracellular DNA sothat the cell-penetrating polypeptide binds extracellular DNA near oraround the live cells so as to form a complex or association therewithsuch that the complex or associated polypeptide-DNA so bound bind thelive cells and penetrates the live cells thereby selectively targetinglive cells at a site of interest with the cell-penetrating polypeptide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. 3E10 scFv penetrates most efficiently into living cellssurrounding a dead cell. GM02605 fibroblasts were washed with serum freemedia and then treated with 10 μM 3E10 scFv for one hour, followed byanti-Myc immunostaining to detect nuclear penetration by 3E10 scFv.Nuclear penetration by 3E10 scFv was restricted to cells in closeproximity to a dead cell, suggesting that a factor released by deadcells promotes nuclear uptake of the fragment.

FIG. 2. Extracellular DNA facilitates penetration of 3E10 scFv into cellnuclei. GM02605 fibroblasts were washed with serum free media and thentreated with control buffer alone or 10 μM 3E10 scFv in the presence ofcontrol buffer, cell lysate, DNA-depleted cell lysate, or purified DNAfor one hour, followed by anti-Myc immunostaining to detect nuclearpenetration by 3E10 scFv. Nuclear penetration into ˜100% of the cellswas only observed in the presence of cell lysate or purified DNA.

FIG. 3. 3E10 scFv localizes to tumor cell nuclei in vivo.Immunodeficient mice bearing subcutaneous U87 human glioma xenograftswere treated by intraperitoneal injection of control buffer or 3E10scFv. Mice were sacrificed 4 or 24 hours after treatment, and tumors andselect normal tissues were immunostained for the presence of 3E10 scFv.(A) Four hours after treatment 3E10 scFv was detected in the nuclei ofthe U87 tumor cells but was not detected in tissues of major organsincluding heart, kidney, skeletal muscle, and liver. These results areconsistent with enhanced uptake of 3E10 scFv into sites of high cellturnover where DNA is released from dying cells. (B) Twenty-four hoursafter treatment 3E10 scFv was still detectable in the tumors,demonstrating the stability of the uptake into tumor nuclei.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that the present disclosure is not limited toparticular embodiments described, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present disclosure will belimited only by the appended claims.

The detailed description of the present disclosure is divided intovarious sections only for the reader's convenience and disclosure foundin any section may be combined with that in another section. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which the present disclosure belongs.

DEFINITIONS

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acompound” includes a plurality of compounds.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the present disclosure belongs. As used herein thefollowing terms have the following meanings.

As used herein, the term “about” when used before a numericaldesignation, e.g., temperature, time, amount, concentration, and suchother, including a range, indicates approximations which may vary by (+)or (−) 10%, 5% or 1%.

As used herein, the term “administration” may be effected in one dose,continuously or intermittently or by several subdoses which in theaggregate provide for a single dose. Dosing can be conducted throughoutthe course of treatment. Methods of determining the most effective meansand dosage of administration are known to those of skill in the art andwill vary with the composition used for therapy, the purpose of thetherapy, the target cell being treated and the subject being treated.Single or multiple administrations can be carried out with the doselevel and pattern being selected by the treating physician. Suitabledosage formulations and methods of administering the agents are known inthe art. Route of administration can also be determined and method ofdetermining the most effective route of administration are known tothose of skill in the art and will vary with the composition used fortreatment, the purpose of the treatment, the health condition or diseasestage of the subject being treated and target cell or tissue.Non-limiting examples of route of administration include intratumoraldelivery, peritumoral delivery, intraperitoneal delivery, intrathecaldelivery, intramuscular injection, subcutaneous injection, intravenousdelivery, nasal spray and other mucosal delivery (e.g. transmucosaldelivery), intra-arterial delivery, intraventricular delivery,intrasternal delivery, intracranial delivery, intradermal injection,electroincorporation (e.g., with electroporation), ultrasound, jetinjector, oral and topical patches.

A “therapeutic agent,” as used herein, may be a molecule, or compoundthat is useful in treatment of a disease or condition. A“therapeutically effective amount,” “therapeutically effectiveconcentration” or “therapeutically effective dose” is the amount of acompound that produces a desired therapeutic effect in a subject, suchas preventing or treating a target condition, alleviating symptomsassociated with the condition, producing a desired physiological effect,or allowing imaging or diagnosis of a condition that leads to treatmentof the disease or condition. The precise therapeutically effectiveamount is the amount of the composition that will yield the mosteffective results in terms of efficacy of treatment in a given subject.This amount will vary depending upon a variety of factors, including,but not limited to, the characteristics of the therapeutic compound(including activity, pharmacokinetics, pharmacodynamics, andbioavailability), the physiological condition of the subject (includingage, sex, disease type and stage, general physical condition,responsiveness to a given dosage, and type of medication), the nature ofthe pharmaceutically acceptable carrier or carriers in the formulation,and the route of administration. One skilled in the clinical andpharmacological arts will be able to determine a therapeuticallyeffective amount through routine experimentation, namely by monitoring asubject's response to administration of a compound and adjusting thedosage accordingly. For additional guidance, see Remington: The Scienceand Practice of Pharmacy 21^((st)) Edition, Univ. of Sciences inPhiladelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, Pa.,2005.

As used herein, “in combination” or “in combination with,” when usedherein in the context of multiple agents, therapeutics, or treatments,means in the course of treating the same disease or condition in asubject administering two or more agents, drugs, treatment regimens,treatment modalities or a combination thereof. This includessimultaneous administration (or “coadministration”), administration of afirst agent prior to or after administration of a second agent, as wellas in a temporally spaced order of up to several days apart. Suchcombination treatment may also include more than a single administrationof any one or more of the agents, drugs, treatment regimens or treatmentmodalities. Further, the administration of the two or more agents,drugs, treatment regimens, treatment modalities or a combination thereofmay be by the same or different routes of administration.

“Treating” or “treatment” of a condition, disease or disorder may referto preventing the condition, disease or disorder, slowing the onset orrate of development of the condition, disease or disorder, reducing therisk of developing the condition, disease or disorder, preventing ordelaying the development of symptoms associated with the condition,disease or disorder, reducing or ending symptoms associated with thecondition, disease or disorder, generating a complete or partialregression of the condition, disease or disorder, or some combinationthereof. Examples of diseases or disorders include colorectal cancer,osteosarcoma, non-small cell lung cancer, breast cancer, ovarian cancer,glial cancer, solid tumors, metastatic tumor, acute lymphoblasticleukemia, acute myelogenous leukemia, adrenocortical carcinoma, Kaposisarcoma, lymphoma, anal cancer, astrocytomas, basal cell carcinoma, bileduct cancer, bladder cancer, bone cancer, brain tumor, breast cancer,bronchial tumor, cervical cancer, chronic lymphocytic leukemia, chronicmyelogenous leukemia, chronic myeloproliferative disorders, coloncancer, colorectal cancers, ductal carcinoma in situ, endometrialcancer, esophageal cancer, eye cancer, intraocular, retinoblastoma,metastatic melanoma, gallbladder cancer, gastric cancer,gastrointestinal carcinoid tumor, gastrointestinal stromal tumors,glioblastoma, glioma, hairy cell leukemia, head and neck cancer,hepatocellular carcinoma, hepatoma, Hodgkin lymphoma, hypopharyngealcancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oralcavity cancer, liver cancer, lobular carcinoma in situ, lung cancer,non-small cell lung cancer, small cell lung cancer, lymphoma,AIDS-related lymphoma, Burkitt lymphoma, non-Hodgkin lymphoma, cutaneousT-cell lymphoma, melanoma, squamous neck cancer, mouth cancer, multiplemyeloma, myelodysplastic syndromes, myelodysplastic/myeloproliferativeneoplasms, nasal cavity and paranasal sinus cancer, nasopharyngealcancer, neuroblastoma, oral cavity cancer, oropharyngeal cancer,osteosarcoma, ovarian cancer, pancreatic carcinoma, papillarycarcinomas, parathyroid cancer, penile cancer, pharyngeal cancer,pheochromocytoma, pineal parenchymal tumors, pineoblastoma, pituitarytumor, pleuropulmonary blastoma, primary central nervous systemlymphoma, prostate cancer, rectal cancer, renal cell cancer, salivarygland cancer, sarcoma, Ewing sarcoma, soft tissue sarcoma, squamous cellcarcinoma, Sezary syndrome, skin cancer, Merkel cell carcinoma,testicular cancer, throat cancer, thymoma, thymic carcinoma, thyroidcancer, urethral cancer, endometrial cancer, uterine cancer, uterinesarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia,and Wilms tumor.

“Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. The terms “cancer”, “cancerous”, “cellproliferative disorder”, “proliferative disorder” and “tumor” are notmutually exclusive as referred to herein. Tumor or cancer status mayalso be assessed by sampling for the number, concentration or density oftumor or cancer cells, alone or with respect to a reference. Inaccordance with the practice of the invention, inhibiting a tumor may bemeasured in any way as is known and accepted in the art, includingcomplete regression of the tumor(s) (complete response); reduction insize or volume of the tumor(s) or even a slowing in a previouslyobserved growth of a tumor(s), e.g., at least about a 10-30% decrease inthe sum of the longest diameter (LD) of a tumor, taking as reference thebaseline sum LD (partial response); mixed response (regression orstabilization of some tumors but not others); or no apparent growth orprogression of tumor(s) or neither sufficient shrinkage to qualify forpartial response nor sufficient increase to qualify for progressivedisease, taking as reference the smallest sum LD since the treatmentstarted (stable disease).

Examples of cytotoxic agents include, but are not limited to ricin,ricin A-chain, doxorubicin, daunorubicin, taxol, ethidium bromide,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine,dihydroxy anthracenedione, actinomycin D, diphtheria toxin, Pseudomonasexotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain,alpha-sarcin, gelonin, mitogellin, restrictocin, phenomycin, enomycin,curcin, crotin, calicheamicin, saponaria officinalis inhibitor,maytansinoids, and glucocorticoid and other chemotherapeutic agents, aswell as radioisotopes such as ²¹²Bi, ¹³¹I, ¹³¹Y and ¹⁸⁶Re. Suitabledetectable markers include, but are not limited to, a radioisotope, afluorescent compound, a bioluminescent compound, chemiluminescentcompound, a metal chelator or an enzyme.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment or man-madeenvironment in which the antibody is synthesized and/or assembled. An“isolated” antibody is deficient in and, preferably, eliminated ofcontaminant components of its natural environment or man-madeenvironment in which the antibody is synthesized and/or assembled.Contaminant components of its natural environment or man-madeenvironment are materials which would interfere with diagnostic ortherapeutic uses for the antibody, and may include enzymes, hormones,and other proteinaceous or nonproteinaceous solutes. “Isolated” antibodydoes not require absolute purity. In some embodiments, the antibody willbe purified (1) to greater than 95% by weight of antibody as determinedby the Lowry method, and most preferably more than 99% by weight, or (2)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Other purificationmethods are well known and contemplated herein.

The term “vector,” is intended to refer to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. Examples of vectors include, but are not limited to, plasmids,(e.g., a circular double stranded DNA loop into which additional DNAsegments may be ligated or introduced), phage vectors, and viral vectors(e.g., wherein additional DNA segments may be ligated or introduced intothe viral genome). Certain vectors are capable of directing theexpression of genes to which they are operatively linked. Such vectorsare referred to herein as “recombinant expression vectors” (or simply,“recombinant vectors”).

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase, or by a syntheticreaction.

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and include monoclonal antibodies (for example, fulllength or intact monoclonal antibodies), polyclonal antibodies,multivalent antibodies, multispecific antibodies (e.g., bispecificantibodies so long as they exhibit the desired biological activity) andmay also include certain antibody fragments (as described in greaterdetail herein). An antibody can be human, humanized and/or affinitymatured.

The term “variable region” or “variable domain” refers to a region ordomain, which is characterized by the presence of certain portions ofthe antibody differing extensively in sequence among antibodies and isused in the binding of each particular antibody to a particular antigen.The “variable region” or “variable domain” confers binding specificityto the antibody. Sequence variability is not evenly distributedthroughout the variable domain or variable region of an antibody.Rather, sequence variability is concentrated in three segments calledcomplementarity-determining regions (CDRs) or hypervariable regions bothin the light-chain and the heavy-chain variable domains. The more highlyconserved portions of variable domains are called the framework (FR).The variable domains of native heavy and light chains each comprise fourFR regions, largely adopting a n-sheet configuration, connected by threeCDRs, which form loops connecting, and in some cases forming part of,the n-sheet structure. The CDRs in each chain are held together in closeproximity by the FR regions and, with the CDRs from the other chain,contribute to the formation of the antigen-binding site of antibodies(see Kabat et al., Sequences of Proteins of Immunological Interest,Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). Theconstant domains are not involved directly in binding an antibody to anantigen, but exhibit various effector functions, such as participationof the antibody in antibody-dependent cellular toxicity.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. In a two-chain Fv species, thisregion consists of a dimer of one heavy- and one light-chain variabledomain in tight, non-covalent association. In a single-chain Fv species,one heavy- and one light-chain variable domain may be covalently linkedby a flexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies.

The Fab fragment contains the constant domain of the light chain and thefirst constant domain (CH1) of the heavy chain. Fab′ fragments differfrom Fab fragments by the addition of a few residues at the carboxyterminus of the heavy chain CH1 domain including one or more cysteinesfrom the antibody hinge region. F(ab′)₂ antibody fragments originallywere produced as pairs of Fab′ fragments which have hinge cysteinesbetween them. Other chemical couplings of antibody fragments are alsoknown.

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion preferably retains at least one, preferably most orall, of the functions normally associated with that portion when presentin an intact antibody. Examples of antibody fragments include Fab, Fab′,F(ab′)₂, and Fv fragments; diabodies; linear antibodies; single-chainantibody molecules; single chain Fv fragment (scFv) and multispecificantibodies formed from antibody fragments. In one embodiment, anantibody fragment comprises an antigen binding site of the intactantibody and thus retains the ability to bind antigen. In anotherembodiment, an antibody fragment, for example, comprises the Fc region,retains at least one of the biological functions normally associatedwith the Fc region when present in an intact antibody, such as FcRnbinding, antibody half-life modulation, ADCC function and complementbinding. In one embodiment, an antibody fragment is a monovalentantibody that has an in vivo half-life substantially similar to anintact antibody. For example, such an antibody fragment may comprise onantigen binding arm linked to an Fc sequence capable of conferring invivo stability to the fragment.

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. Humanized antibodies may be human immunoglobulins(recipient antibody) in which residues from a hypervariable region ofthe recipient are replaced by residues from a hypervariable region of anon-human species (donor antibody) such as mouse, rat, rabbit ornonhuman primate having the desired specificity, affinity, and capacity.The humanized antibody may optionally also comprise at least a portionof an immunoglobulin constant region (Fc), e.g. that of a humanimmunoglobulin.

“Chimeric” antibodies have a portion of the heavy and/or light chainidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (Morrison et al., Proc. Natl. Acad. Sci. USA81:6851-6855 (1984)).

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For example, oneembodiment of 3E10 mAb is a 3E10 scFv having the primary structure shownin FIG. 4-1 through 4-4 of U.S. Patent Application Publication No.: US2013/0266570 A1, published 10 Oct. 2013.

An “antigen” is a target to which an antibody can selectively bind. Thetarget antigen may be polypeptide, carbohydrate, nucleic acid, lipid,hapten or other naturally occurring or synthetic compound.

In accordance with the practice of the invention, “extracellular DNA” isDNA free of a cell or cell-free DNA. Extracellular DNA may be introducedor administered by methods known in the art such as, for example,microinjection of DNA into extracellular space or outside of a cell orcells, cell or cells in a tissue, or cell or cells in an organ, so longas the DNA is not introduced or administered into a cell. The deliveredDNA in the extracellular space or outside of a cell may be in anyphysical state, including in a solution, as a solid, as a colloidalform, as a semi-crystalline state, as a nanoparticle or combinationthereof. The delivered DNA may be isolated DNA from nature, eithertotal, fractionated, intact or sheared, or synthesized by the hand ofman such as through synthetic chemistry or in vitro enzymatic methods asknown in the art. Alternatively, the extracellular DNA may be producedin situ by dying cells that release its nucleic acid content. In oneembodiment, extracellular DNA is produced by radiation, chemotherapeuticagent, toxin, or by any condition that promotes cell death. In oneembodiment, the invention contemplates the use of radiation or achemotherapeutic agent at a site of interest or a site of injury toproduce extracellular DNA or additional extracellular DNA. In anotherembodiment, extracellular DNA may be produced in situ through freezing,heat, laser, hypoxic condition, a poison, laceration, force and trauma.

A “bispecific antibody” of the invention includes antibodies with notonly binding specificities for two targets but also include antibodieswith additional determinants, which may be derived from immunoglobulinsequences or non-immunoglobulin sequences, with specificities for othertarget(s). For example, a bispecific antibody includes heteroconjugateswith binding specificities for at least two different targets. Forexample a heteroconjugates includes a hybrid antibody created fromlinking two different antibodies or antibody fragments or a hybrid of anantibody or antibody fragment linked to a lectin or lectin fragment oranother determinant with an intracellular binding specificity or acell-penetrating ability, so long as the heteroconjugates have bindingspecificities for at least two targets. A bispecific antibody mayfurther include heteroconjugates in which a bispecific antibody iscoupled to a therapeutic agent (e.g., chemotherapeutic agent or toxin orcytoprotective agent) or an imaging agent (e.g., radioisotope). Abispecific antibody may be produced by recombinant DNA methods in whichcoding sequences of immunoglobulin genes are manipulated to produce thebispecific antibody. The coding sequences of the immunoglobulin genesmay be used in its entirety, mutated at specific sequences or codons, orused partially by truncating the coding sequences to produce thebispecific antibody or components that results in production of abispecific antibody. In some embodiments, a bispecific antibody includesan intact antibody or a Fv fragment, Fab, Fab′ or F(ab′)₂ fragment or adiabody, linear antibody, single-chain antibody molecule or scFvantibody fragment coupled chemically or recombinantly, disulfide bridgesor by other means to a second determinant which specifically recognizesat least a different target than the target recognized by the intactantibody or the Fv, Fab, Fab′ or F(ab′)₂ fragment or the diabody, linearantibody, single-chain antibody molecule or scFv antibody fragment. Thesecond determinant includes a second intact antibody or a Fv fragment,Fab, Fab′ or F(ab′)₂ fragment or a diabody, linear antibody,single-chain antibody molecule or scFv antibody fragment different fromthe binding specificity of the first antibody or the first Fv, Fab, Fab′or F(ab′)₂ fragment or a the first diabody, linear antibody,single-chain antibody molecule or scFv antibody fragment.

In accordance with the practice of the invention, the second determinantmay recognize a target that is located inside the cell, e.g., in thecytoplasm or in the cell nucleus. In another embodiment, the seconddeterminant recognizes a target that is normally an intracellularprotein and not normally on the surface of a cell or not normallysecreted by the cell. In a further embodiment, the second determinantrecognizes an E3 ubiquitin-protein ligase, a tumorsuppressor-interacting protein, a binding partner of a tumor suppressorprotein, an oncoprotein, or a DNA repair protein, wherein the seconddeterminant fails to recognize any protein that normally resides on thecell surface. In yet a further embodiment, the second determinantrecognizes a transcription factor, a transcriptional repressor, atranscriptional co-factor, a nuclear receptor, a steroid receptor, amethylase, an acetylase, a deacetylase, RNA polymerase, a kinase, aphosphatase, an intracellular signaling molecule (not a cell surfacesignaling molecule), a cell cycle regulatory protein, a protease, a DNArepair protein, a recombinase, a chromosomal protein, an apoptoticprotein, a SUMO ligase, a ubiquitin ligase, a metabolic protein, anorganelle protein, a nuclear protein, a nucleolar protein, amitochondrial protein, a ligand, a ribosomal protein, an enzyme, acytoskeletal protein, a chromosomal protein, a structural protein, aintracellular soluble protein, an intracellular shuttling protein or aregulatory protein so long as the second determinant fails to recognizeany protein that normally resides on the cell surface.

A bispecific antibody includes chimeric antibodies, recombinantantibodies, humanized antibodies or human antibodies or theirderivatives. A bispecific antibody includes antibodies of the inventionin which one or more of the complementarity determining region (CDR) ofthe invention is used to screen for additional antibodies or agents thatcan compete with the binding of the 3E10 antibody. Peptide, phagedisplay, cDNA, or chemical libraries may be used for such a screen.

Examples of 3E10 bispecific antibodies, e.g., 3E10 scFv-3G5 scFvbispecific antibody and 3E10 scFv-PAb421 scFv bispecific antibody, aredisclosed in U.S. Ser. No. 13/844,318, filed Mar. 15, 2013, which isincorporated by reference herein. Additional examples of anti-DNAmonoclonal antibody 3E10 (also referred to herein as a 3E10 antibody ormAb 3E10) include an antibody produced by ATCC PTA 2439 or a functionalfragment or variant thereof or an antibody having the specificity of mAb3E10 (Chan G, et al., Int. J. Cancer 2016 138(1):182-6; Weisbart R H, etal., Sci. Rep. 2015 5:12022; Noble P W, et al., Cancer Res. 201575(11):2285-91; Hansen J E, et al., Sci. Transl. Med. 2012 24;4(157):157ra142; Weisbart R H, et al., Mol. Cancer Ther. 201211(10):2169-73; Heinze E, et al., Oncol. Lett. 2011 2(4):665-668; ZhanX, et al., Stroke. 2010 41(3):538-43; Hansen J E, et al., J. Biol. Chem.2007 282(29):20790-3; Hansen J E, et al., Cancer Res. 200767(4):1769-74; Hansen J E, et al., Brain Res. 2006 1088(1):187-96;Hansen J E, et al., Scientific World Journal. 2005 5:782-8; Weisbart RH, et al., J. Drug Target. 2005 13(2):81-7; Weisbart R H, et al., Int.J. Oncol. 2004 25(6):1867-73; Weisbart R H, et al., Int. J. Oncol. 200425(4):1113-8; Weisbart R H, et al., Cancer Lett. 2003 195(2):211-9;Weisbart R H, et al., Mol. Immunol. 2003 39(13):783-9; Weisbart R H, etal., J. Immunol. 2000 164(11):6020-6; Spertini F, et al., J Rheumatol.1999 26(12):2602-8; Weisbart R H, et al., J. Autoimmun. 199811(5):539-46; Zack D J, et al., J. Immunol. 1996 157(5):2082-8; Zack DJ, et al., Mol. Immunol. 1995 32(17-18):1345-53; Zack D J, et al., J.Immunol. 1995 154(4):1987-94; Zack D J, et al., Immunol. Cell Biol. 199472(6):513-20; and Weisbart R H, et al., J. Immunol. 1990 144(7):2653-8).The full 3E10 antibody has been previously described (Weisbart R H, etal., J. Immunol. 1990 144(7): 2653-2658; ATCC Accession No. PTA 2439hybridoma; SEQ ID NOS.: 2 and 4 of PCT International Publication No.: WO2010/148010 A1, published 23 Dec. 2010) as well as its nucleic acidsequence and protein sequence (FIGS. 3 and 4 of Zack D J, et al., J.Immunol. 1995 154(4): 1987-1994; FIGS. 3 and 4 of US Patent ApplicationPublication No.: US 2008/0292618 A1; FIGS. 1 and 2 of PCT InternationalPublication No.: WO 2010/138769 A1, published 2 Dec. 2010; GenBankAccession Numbers: L16982 for mAb 3E10 VH chain and L34051 for mAb 3E10Vκ light chain). Location of the complement-determining regions (e.g.,CDR1, CDR2 and CDR3) along with the framework regions (i.e., FR1, FR2,FR3, and FR4) of the 3E10 variable heavy chain and light chain domainsare provided in FIGS. 3 and 4 of Zack D J, et al., J. Immunol. 1995154(4): 1987-1994; FIGS. 3 and 4 of US Patent Application PublicationNo.: US 2008/0292618 A1; FIGS. 1 and 2 of PCT International PublicationNo. WO 2010/138769 A1, published 2 Dec. 2010. Particularly usefulvariant is substitution of aspartic acid (D) at amino acid position 31of the heavy chain variable region (VH) of 3E10 antibody with asparagine(N), the D31N variant, which increases binding to ssDNA and dsDNA (ZackD J, et al., J. Immunol. 1995 154(4): 1987-1994) and enhances cell andnuclear penetration (Zack D J, et al., J. Immunol. 1996 157(5):2082-2088; Weisbart W H, et al., J. Autoimmunity 11(5): 539-546). Apreferred embodiment of 3E10 antibody or its fragment is 3E10 antibodyor its fragment or derivative with aspartic acid to asparagine change atamino acid 31 of the VH chain, the D31N variant.

A “subject” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, farm animals (such ascows), pets (such as cats, dogs and horses), primates, mice and rats.

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic orprophylactic result.

A “therapeutically effective amount” of a substance/molecule of theinvention, agonist or antagonist may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the substance/molecule, agonist or antagonist to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of thesubstance/molecule, agonist or antagonist are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typicallybut not necessarily, since a prophylactic dose is used in subjects priorto or at an earlier stage of disease, the prophylactically effectiveamount will be less than the therapeutically effective amount.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes,chemotherapeutic agents e.g. methotrexate, adriamycin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents, enzymes andfragments thereof such as nucleolytic enzymes, antibiotics, and toxinssuch as small molecule toxins or enzymatically active toxins ofbacterial, fungal, plant or animal origin, including fragments and/orvariants thereof, and the various antitumor or anticancer agentsdisclosed below. Other cytotoxic agents are described below. Atumoricidal agent causes destruction of tumor cells.

According to the present invention, where administration includes apharmaceutical formulation, preferably the formulation is a unit dosagecontaining a daily dose or unit, daily sub-dose or an appropriatefraction thereof, of the active ingredient (also referred to herein as atherapeutic agent). In one embodiment, the active ingredient comprises acell-penetrating polypeptide of the invention. In one embodiment, theactive ingredient comprises a cell-penetrating polypeptide-conjugate,such as a cell-penetrating polypeptide chemically crosslinked to achemical, peptide or protein with a desired biological activity, acell-penetrating polypeptide modified with a radioisotope or modifiedwith a chelator bound to a radioisotope, or a cell-penetratingpolypeptide linked to a peptide or protein (and produced) throughrecombinant DNA methods. In another embodiment, the active ingredientcomprises a fusion protein of a cell-penetrating polypeptide and asecond protein or peptide with a desired biological activity. In oneembodiment, the desired biological activity may be an activity thatinduces cell death or is cell protective. In one embodiment, the activeingredient is DNA and/or its degradation product(s). In one embodiment,the active ingredient is extracellular DNA and/or its degradationproduct(s).

The compositions of the invention can be administered by any parenteralroute, in the form of a pharmaceutical formulation comprising the activeingredient, optionally in the form of a nontoxic organic, or inorganic,acid, or base, addition salt, in a pharmaceutically acceptable dosageform. Depending upon the disorder and patient to be treated, as well asthe route of administration, the compositions may be administered atvarying doses.

In human therapy, compositions of the invention may be administeredalone but may generally be administered in admixture with a suitablepharmaceutical excipient diluent or carrier selected with regard to theintended route of administration and standard pharmaceutical practice.

In embodiments of the present invention in which polypeptides orpolynucleotides of the invention are administered parenterally, suchadministration can be, for example, intravenously, intra-arterially,intraperitoneally, intrathecally, intraventricularly, intracisternally,intracranially, intramuscularly or subcutaneously, or they may beadministered by infusion techniques. They are best used in the form of asterile aqueous solution which may contain other substances, forexample, enough salts or glucose to make the solution isotonic withblood. The aqueous solutions should be suitably buffered (preferably toa pH of from 3 to 9), if necessary. The preparation of suitableparenteral formulations under sterile conditions is readily accomplishedby standard pharmaceutical techniques well-known to those skilled in theart.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Methods of the Invention

The invention is directed to the discovery that certain anti-DNAantibodies, including but not limited to, 3E10 monoclonal antibody orother anti-DNA antibodies that by their dependence on a salvage pathwayfor cell and nuclear penetration, require extracellular DNA or itsdegradation product in order to penetrate cells in a salvagepathway-dependent process, such as a nucleoside salvage pathway or ENT-2nucleoside transporter pathway. This requirement for extracellular DNAor its degradation product for cell and nuclear penetration provides anopportunity for “targeted” therapies with these anti-DNA antibodies byintroducing or producing extracellular DNA or its degradation productnear or around live cells at a site of interest being targeted by thesecell-penetrating anti-DNA antibodies. “Targeted” therapies, using suchanti-DNA antibodies or their conjugates, reduce any potential systemictoxicity as well as increase effectiveness of the antibodies and theirconjugates.

In one embodiment, the invention provides a method for selectivetargeting of live cells at a site of interest with a cell-penetratingpolypeptide which comprises: (a) introducing or producing extracellularDNA and/or its degradation product(s) near or around the live cells atthe site of interest; (b) introducing the cell-penetrating polypeptidecomprising cell-penetrating determinants near or around the live cells,before, after or concurrently with the DNA of step (a); (c) contactingextracellular DNA or its degradation product near or around the livecells with a cell-penetrating polypeptide comprising cell-penetratingdeterminants so that the cell-penetrating polypeptide bindsextracellular DNA or its degradation product near or around the livecells so as to form a complex; (d) contacting one of the live cells withthe complex in (c) so as to bind and penetrate the live cell; and (e)permitting additional complexes to form as in (c) and contactingadditional cells with said complexes so as to bind and penetrateadditional live cells at the site of interest; thereby selectivelytargeting live cells at the site of interest with a cell-penetratingpolypeptide.

In an embodiment of the invention, the extracellular DNA and/or itsdegradation product(s) is introduced or produced near or around the livecells at about less than 20 mm from the cells at the site of interest;less than 10 mm from cells at the site of interest; a range of between0.5 mm to 5 mm from the cells at the site of interest; a range ofbetween 0.5 mm to 20 mm from the cells at the site of interest; range ofbetween 0.5 mm to 0.1 mm from the cells at the site of interest; rangeof less than 0.1 mm from the cells at the site of interest; range ofbetween 100 μm to 10 μm from the cells at the site of interest; ordirectly into the site of interest (e.g., directly into the tumor massor cancer). In another embodiment, the extracellular DNA and/or itsdegradation product(s) is introduced near or around the live cells inclose proximity to a target cell, tissue or injury.

The invention provides methods for selective targeting of live cells,which have undergone or are undergoing radiation or chemotherapy, at asite of interest with a cell-penetrating polypeptide. In one embodimentof the invention, the method comprises contacting the live cells with acell-penetrating polypeptide comprising cell-penetrating determinants sothat the cell-penetrating polypeptide binds extracellular DNA near oraround the live cells so as to form a complex or association therewithsuch that the complex or associated polypeptide-DNA so bound binds thelive cells and penetrates the live cells thereby selectively targetinglive cells at a site of interest with a cell-penetrating polypeptide.Examples of cell-penetrating polypeptides include cell-penetratingantibodies such as 3E10, 5C5, 5C6 and 4H2 (Weisbart R H, et al., J.Immunol. 1990 144(7): 2653-2658; Zack D J, et al., J. Immunol. 1995154(4): 1987-1994; Weidle U H, et al., Cancer Genomics Proteomics 201310: 239-250; Weisbart R H, et al., Sci. Rep. 2015 5: 12022; Noble P W,et al., Sci. Rep. 2014 4:5958; Colburn K K, et al., J. Rheumatol. 200330(5):993-7). Examples of cell-penetrating determinants include, but arenot limited to, cell-penetrating determinants from antibodies such as3E10, 5C5, 5C6 and 4H2.

In one embodiment, the site of interest is an injury site. Examples ofinjury sites include but are not limited to an intracranial injury,brain injury, heart injury (e.g., myocardial infarction), skin injury,liver injury, gastrointestinal injury, lung injury, eye injury, kidneyinjury, pancreas injury, peritoneal injury, bone injury, nasopharyngealinjury, uterine injury, cervical injury, breast injury, organ injury,tissue injury, burn or radiation injury. An injury may include acellular injury involving tissue or organ injury. Examples of cellularinjury include any of chemical injury, excess reactive oxygen species,burn, hypothermia, ischemia, hypoxia, blunt force trauma, stress, heatshock, cold shock, hypothermia, mechanical stress, hypoxia, ischemia,cellular swelling, DNA damage, DNA fragmentation, membrane damage,organelle damage, damage due to heat, damage due to cold, damage due toradiation, damage due to chemical exposure, damage due to dehydration,mitochondrial damage, activation of apoptotic pathway, damage due to aninfection, damage due to acidification, damage due to proteinmisfolding, damage due to intracellular protein aggregation, damage dueto laser, damage due to aspiration, damage due to vacuum, damage due tocellular stress, injury due to damage to cell membrane, damage due tochanges in osmotic pressure, or any cellular malfunction that results incell death or results in altered cell proliferation that is deleterious,such as a cancer or other diseases or disorders referred to herein.

Additionally, the invention provides method for selective targeting oflive cells at or near the proximity of a cellular injury with acell-penetrating polypeptide which comprises cell-penetratingdeterminants which polypeptide is optionally joined to, or combinedwith, a therapeutic agent. In one embodiment, the method comprisingadministering the cell-penetrating polypeptide at or near the proximityof the injury so that it binds extracellular DNA from the cellularinjury so as to form a complex or association therewith such that thecomplex or associated polypeptide-DNA-therapeutic agent so bound bindthe live cells and penetrates the live cells thereby selectivelytargeting live cells at a site of interest with the cell-penetratingpolypeptide.

In one embodiment, introducing the cell-penetrating polypeptidecomprising cell-penetrating determinants before, after or concurrentlywith the DNA of step (a) is at a site other than near or around the livecells at the site of interest, such as the introduction of thecell-penetrating polypeptide via an intravenous injection to permitsystemic circulation of the introduced cell-penetrating polypeptide.

The invention also provides methods for inhibiting cellular injury in asubject. In one embodiment the method comprises administering directlyto the live cells at or near a site of cellular injury of the subject acell-penetrating polypeptide comprising cell-penetrating determinantsjoined to a therapeutic agent. The method further comprises the step ofcontacting extracellular DNA or its degradation product with thecell-penetrating polypeptide so that the cell-penetrating polypeptidebinds extracellular DNA or its degradation product near or around thelive cells so as to form a complex. The live cell and the complex comein contact so that the complex can penetrate the live cell. Additionalcomplexes are permitted to form and contact additional cells at the siteof injury so that that the complexes penetrate additional live cells atthe site of cellular injury; thereby, inhibiting cellular injury in thesubject.

The invention also provides methods for inhibiting a cell or inducingcell death in a subject comprising administering directly to the livecells at or near a site of injury of the subject a cell-penetratingpolypeptide comprising cell-penetrating determinants which polypeptideis optionally joined to a therapeutic agent. The method furthercomprises the step of contacting extracellular DNA or its degradationproduct near or around the live cells with the cell-penetratingpolypeptide so that the cell-penetrating polypeptide binds extracellularDNA or its degradation product near or around the live cells therebyforming a complex which can bind and penetrate the live cell whichinduces cell death or inhibition. Additional complexes are permitted toform and contact additional cells at the site of injury so that that thecomplexes penetrate additional live cells live cells at the site ofinjury thereby inducing cell death or inhibition at or near a site ofinjury with the cell-penetrating polypeptide.

In one embodiment, “introducing or producing extracellular DNA and/orits degradation product(s) near or around the live cells at the site ofinterest” comprises administering DNA and/or its degradation product(s).In one embodiment, DNA is administered into the extracellular space oroutside of a cell (e.g., not inside a cell) by any method known in theart, including injection, microinjection, microprojectile andimplantation. In another embodiment, “introducing, producing orpermitting presence of extracellular DNA and/or its degradationproduct(s) near or around the live cells at the site of interest”comprises a man-made intervention to produce localized cellular damage,such as through radiation (e.g., low dose radiation), chemotherapeuticagent, cytotoxic drug, toxin, hypoxia, blunt force trauma, hypothermia,burn or an infectious agent, so as to cause cell death and release ofchromosomal DNA.

In one embodiment, the cell-penetrating polypeptide is administered orintroduced at a site of interest. In another embodiment, thecell-penetrating polypeptide is administered or introduced (e.g.,directly administered or introduced) near or around a site of interest.In an embodiment of the invention, a site of interest is a site ofinjury, e.g., cellular injury. In another embodiment, the cellpenetrating polypeptide is administered or introduced at a site ofcellular injury. In another embodiment, the cell-penetrating polypeptideis administered or introduced at a site away from the injury. Merely byway of example, the cell-penetrating polypeptide may be administered orintroduced in a subject by subcutaneous injection, intramuscularinjection or intravenous injection. In another example, thecell-penetrating polypeptide is administered or introduced to a subjectwherein the cell-penetrating polypeptide circulates systemically.

In one embodiment, the extracellular DNA is administered or introducedat a site of interest. It may be coadministered with thecell-penetrating polypeptide. Alternatively, the extracellular DNA maybe separately administered with the cell-penetrating polypeptide, e.g.,administered before or after the administration of the cell-penetratingpolypeptide. In one embodiment of the invention, the extracellular DNAis administered or introduced at a site of injury. In anotherembodiment, the extracellular DNA is administered or introduced at asite of cellular injury. In one embodiment, the extracellular DNA isadministered or introduced (e.g., directly administered or introduced)near or around the live cells at a site of interest, e.g., a site ofinjury. In another embodiment, extracellular DNA is administered orintroduced near or around the live cells at a site of cellular injury.In one embodiment, the extracellular DNA may be produced in situ throughthe use of cell damaging agents. In one embodiment, the extracellularDNA is produced by the act of man at a site of interest in a subject.

In another embodiment, the invention provides a method for inhibitingcellular injury in a subject comprising: (a) administering directly tothe live cells at or near a site of cellular injury of the subject acell-penetrating polypeptide comprising cell-penetrating determinantsjoined to a therapeutic agent; (b) contacting extracellular DNA or itsdegradation product with the cell-penetrating polypeptide so that thecell-penetrating polypeptide binds extracellular DNA or its degradationproduct near or around the live cells so as to form a complex; (c)contacting one of the live cells with the complex in (b) so as to bindand penetrate the live cell; and (d) permitting additional complexes toform as in (b) and contacting additional cells with said complexes so asto bind and penetrate additional live cells at the site of cellularinjury; thereby, inhibiting cellular injury in the subject

In a further embodiment, the invention provides a method for selectivetargeting of live cells at or near a site of cellular injury with acell-penetrating polypeptide which comprises cell-penetratingdeterminants joined to a therapeutic agent, the method comprising: (a)contacting the live cells with a composition comprising (i) acell-penetrating polypeptide which comprises cell-penetratingdeterminants joined to a therapeutic agent and (ii) extracellular DNA orits degradation product so that the cell-penetrating polypeptide bindsextracellular DNA or its degradation product near or around the livecells so as to form a complex such that the complex so formed binds oneof the live cells and penetrates the live cell; and (b) permittingadditional complexes to form as in (a) and contacting additional cellswith said complexes so as to bind and penetrate additional live cells atthe site of cellular injury, thereby selectively targeting live cells ata site of cellular injury with a cell-penetrating polypeptide.

In yet a further embodiment, the invention provides a method forinducing cell death in a subject comprising: (a) administering directlyto the live cells at or near a site of injury of the subject acell-penetrating polypeptide comprising cell-penetrating determinantsjoined to a therapeutic agent; (b) contacting extracellular DNA or itsdegradation product near or around the live cells with thecell-penetrating polypeptide so that the cell-penetrating polypeptidebinds extracellular DNA or its degradation product near or around thelive cells so as to form a complex; (c) contacting one of the live cellswith the complex in (b) so as to bind and penetrate the live cell whichinduces cell death; and (d) permitting additional complexes to form asin (b) and contacting additional cells with said complexes so as to bindand penetrate additional live cells inducing additional cell death atthe site of injury; thereby inducing cell death at or near a site ofinjury with the cell-penetrating polypeptide.

In yet another embodiment, the methods of the invention further providesthe step of administering DNA and/or its degradation product (s) to theinjury site or to a site of interest in an extracellular space tofacilitate further selective targeting. In one embodiment, theintroduced or administered DNA is a double-stranded DNA. In anotherembodiment, the introduced or administered DNA is a single-stranded DNA.In one embodiment, the introduced or administered DNA is isolated orpurified DNA isolated from a cell (e.g., DNA from a cell lysate orpurified from a cell), a virus or a bacteriophage. In an embodiment ofthe invention, the introduced or administered DNA may have modifiedbases (e.g., 5-methyl-cytosine). In an embodiment of the invention, theintroduced or administered DNA is synthesized DNA such as a chemicallysynthesized oligonucleotide with or without modified bases. In anembodiment of the invention, the DNA so introduced or administered isnot purified away from non-DNA nucleic acid (e.g., RNA), nucleotide,nucleoside or purine or pyrimidine base. In an embodiment of theinvention, the DNA so introduced or administered is purified away fromnon-DNA nucleic acid, nucleotide, nucleoside or purine or pyrimidinebase.

In an embodiment of the invention, the administered DNA is a polymer ofthymidine monophosphate (dTMP) or poly (dT). In a further embodiment,the administered DNA comprises a thymidine or dT or thymine-deoxyribose.For example, in one embodiment a 3E10 antibody or fragment thereof has ahigher binding affinity to single stranded DNA comprising a dT in theDNA sequence. As a further example, the single stranded DNA sointroduced or administered comprises a poly (dT) sequence.

The DNA may be single-stranded DNA having a length of about 40,000 basesto about 2 bases or dinucleotide. In a further embodiment, the DNA mayhave a length of about more than 1,000 bases. In another embodiment, theDNA may have a length of about more than 2000 bases. In yet anotherembodiment, the DNA may have a length of about less than 2000 bases. Inanother embodiment, the DNA may have a length of about 50 bases to 500bases. In yet another embodiment, the DNA may have a length of aboutless than 100 bases.

The DNA may be double stranded DNA having a length of about less than 20kilobase pairs (kb pairs) to about 5 bp. In one embodiment, the DNA mayhave a length of about less than 2000 base pair (bp). In one embodiment,the DNA may have a length of about more than 2000 base pair (bp). Inanother embodiment, the DNA may have a length of about 50 bp to 500 bp.In yet another embodiment, the DNA may have a length of about less than100 bp. In one embodiment, the DNA is purified calf thymus doublestranded DNA sheared to an average length of 2000 bp.

In one embodiment, the DNA is non-infectious DNA (e.g., complete viralgenome) or enzymatic DNA (e.g., DNAzyme). Preferably, the DNA isnon-infectious DNA.

In one embodiment, the DNA is partially degraded DNA in the DNA-antibodycomplex or DNA-cell-penetrating polypeptide complex before cellpenetration by the antibody or cell-penetrating polypeptide.

In one embodiment, the DNA and/or its degradation product(s) may besingle-stranded, double-stranded, triple-stranded, or four-stranded or acombination thereof.

In an embodiment of the invention, the dose of the DNA and/or itsdegradation product(s) so introduced or administered may be betweenabout 100 μg to 1 μg, between about 1 μg to 100 ng DNA, between about100 ng to 10 ng DNA, between about 10 ng to 1 ng DNA, between about 1 ngto 100 pg DNA, between about 100 pg to 10 pg DNA, or between 10 pg to 1pg DNA. In one embodiment, the dose is less than 1 ng DNA. In yet afurther embodiment, the dose may be may be less than about 100 pg DNA.Factors to be considered in choice of actual dose include condition andtissue/organ being treated, number of cells at the site of interest tobe treated, size of the tumor, size of the injury or ischemia,diffusibility of DNA and/or its degradation product(s) from the site ofinterest, tumor, injury site or ischemic site, and volume of theextracellular space at and around the site of interest. Doses will needto be adjusted based on the desired outcome. Multiple doses may need tobe introduced or administered.

In one embodiment, the dose of the DNA and/or its degradation product(s)is DNA. In another embodiment, the dose of the DNA and/or itsdegradation product(s) is its degradation product(s). In one embodiment,the DNA degradation product(s) may be directly obtained from degradationDNA. In another embodiment, the DNA degradation product(s) may beequivalent to the degradation product(s) directly produced from the DNA.In another embodiment, the DNA degradation product(s) nucleotide,nucleoside, pyrimidine base and/or purine base. In another embodiment,the DNA degradation product(s) is produced in situ from the introducedor administered DNA. In yet another embodiment, the dose of the DNAand/or its degradation product(s) is or comprises both DNA and itsdegradation product(s).

The DNA and/or its degradation product(s) introduced or administered maybe in a liquid formulation or a solid formulation. In one embodiment ofa liquid formulation, the liquid formulation comprises DNA and/or itsdegradation product(s) and an aqueous carrier, preferably isotonic, at aDNA and/or its degradation product(s) concentration of less than 20mg/ml, a DNA and/or its degradation product(s) concentration between 10mg/ml to 1 mg/ml, a DNA concentration between 1 mg/ml to 100 μg/ml, aDNA and/or its degradation product(s) concentration between 100 μg/ml to10 μg/ml, a DNA and/or its degradation product(s) concentration between10 μg/ml to 1 μg/ml, a DNA and/or its degradation product(s)concentration between 1 μg/ml to 100 ng/ml, a DNA and/or its degradationproduct(s) concentration between 100 ng/ml to 10 ng/ml, or a DNA and/orits degradation product(s) concentration between 10 ng/ml to 1 ng/ml. Inone embodiment, the DNA and/or its degradation product(s) concentrationis less than 1 μg/ml. In one embodiment, the DNA and/or its degradationproduct(s) concentration is greater than 1 μg/ml. In a solid formation,the DNA and/or its degradation product(s) may be in the form of freeacid or preferably in a salt. In one embodiment, the solid formulationis an immediate-release formulation comprising DNA and/or itsdegradation product(s) and a suitable carrier. In one embodiment, thesolid formulation is a sustained-release formulation comprising DNAand/or its degradation product(s) and a suitable carrier. In oneembodiment, the solid formulation is a combination of animmediate-release formulation and sustained-release formulation.

In one embodiment, the liquid formulation comprises a cell-penetratingpolypeptide and DNA and/or its degradation product(s). In oneembodiment, the solid formulation comprises a cell-penetratingpolypeptide and DNA and/or its degradation product(s). In a furtherembodiment, the cell-penetrating polypeptide and DNA is in acell-penetrating polypeptide-DNA complex or association. In anotherembodiment, the cell-penetrating polypeptide and DNA degradationproduct(s) is in a cell-penetrating polypeptide-DNA degradation productcomplex or association.

For example, in one embodiment, a site of interest is an injury sitethat may be created through the use of a cell-damaging agent. Examplesof cell-damaging agents include, but are not limited to, a radioisotope,cytotoxic agent or radiation. Suitable examples of radioisotope include⁴⁶Sc, ⁴⁷Sc, ⁴⁸Sc, ⁶⁷Cu, ⁶⁷Ga, ⁷²Ga, ⁷³Ga, ⁹⁰Y, ⁶⁷Cu, ¹⁰⁹Pd, ¹¹¹Ag,¹¹¹In, ¹²⁵, ¹³¹I, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At,²¹¹Bi, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²¹⁴Bi and ²²⁵Ac. Suitable cytotoxic agentsinclude ricin, ricin A-chain, doxorubicin, daunorubicin, paclitaxel,taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicine, dihydroxy anthracenedione, actinomycin D,diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin Achain, modeccin A chain, alpha-sarcin, gelonin, mitogellin,restrictocin, phenomycin, enomycin, curcin, crotin, calicheamicin,saponaria officinalis inhibitor, maytansinoids, and glucocorticoid.Examples of radiation include microwave, infrared, ultraviolet, X-ray,gamma ray, alpha particle radiation, beta ray and ionizing radiation.

In one embodiment, the injury site is created through the use of acell-damaging agent wherein the cell-damaging agent is a DNA damagingagent. In one embodiment, the DNA damaging agent is doxorubicin. In oneembodiment, the injury site is created through the use of acell-damaging agent wherein the cell damaging agent is a DNA damagingagent other than doxorubicin. In one embodiment, the injury site iscreated through the use of a cell-damaging agent other than aDNA-damaging agent. In one embodiment, the injury site is createdthrough the use of a cell-damaging agent other than a DNA-damaging agentis taxol.

In one embodiment, the injury site is created through the use ofradiation. Examples of radiation include microwave, infrared,ultraviolet, X-ray, gamma ray, alpha particle radiation, beta ray andionizing radiation. In one embodiment, the injury site is createdthrough means other than through the use of radiation. Examples ofradiation include microwave, infrared, ultraviolet, X-ray, gamma ray,alpha particle radiation, beta ray and ionizing radiation.

In one embodiment, the site of interest comprises extracellular DNA. Inanother embodiment, the site of interest is devoid or substantiallydevoid of extracellular DNA. In one embodiment, the site of interest isa site within an in vitro cell or organ culture. In one embodiment, thesite of interest is an in vivo site. In one embodiment, the site ofinterest is a site within a subject or a mammal.

The invention further provides methods for selective targeting of livecells at or near the proximity of a cellular injury with acell-penetrating polypeptide. In an embodiment of the invention, thecell-penetrating determinants are joined to, or combined with (e.g., asan admix), a therapeutic agent. The method may comprise contacting thelive cells with a composition having (a) a cell-penetrating polypeptidewhich comprises cell-penetrating determinants and (b) extracellular DNAso that the cell-penetrating polypeptide binds extracellular DNA near oraround the live cells so as to form a complex or association therewithsuch that the complex or associated polypeptide-DNA so bound bind thelive cells and penetrate the live cells thereby selectively targetinglive cells at a site of interest with the cell-penetrating polypeptide.

The invention also provides method for inhibiting a tumor associatedwith ischemia, cellular/tissue necrosis or cellular/tissue apoptosis byselective targeting of live cells at a site of interest by any of themethods of the invention.

Examples of tumors include colorectal cancer, osteosarcoma, non-smallcell lung cancer, breast cancer, ovarian cancer, glial cancer, solidtumors, metastatic tumor, acute lymphoblastic leukemia, acutemyelogenous leukemia, adrenocortical carcinoma, Kaposi sarcoma,lymphoma, anal cancer, astrocytomas, basal cell carcinoma, bile ductcancer, bladder cancer, bone cancer, brain tumor, breast cancer,bronchial tumor, cervical cancer, chronic lymphocytic leukemia, chronicmyelogenous leukemia, chronic myeloproliferative disorders, coloncancer, colorectal cancers, ductal carcinoma in situ, endometrialcancer, esophageal cancer, eye cancer, intraocular, retinoblastoma,metastatic melanoma, gallbladder cancer, gastric cancer,gastrointestinal carcinoid tumor, gastrointestinal stromal tumors,glioblastoma, glioma, hairy cell leukemia, head and neck cancer,hepatocellular carcinoma, hepatoma, Hodgkin lymphoma, hypopharyngealcancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oralcavity cancer, liver cancer, lobular carcinoma in situ, lung cancer,non-small cell lung cancer, small cell lung cancer, lymphoma,AIDS-related lymphoma, Burkitt lymphoma, non-Hodgkin lymphoma, cutaneousT-cell lymphoma, melanoma, squamous neck cancer, mouth cancer, multiplemyeloma, myelodysplastic syndromes, myelodysplastic/myeloproliferativeneoplasms, nasal cavity and paranasal sinus cancer, nasopharyngealcancer, neuroblastoma, oral cavity cancer, oropharyngeal cancer,osteosarcoma, ovarian cancer, pancreatic carcinoma, papillarycarcinomas, parathyroid cancer, penile cancer, pharyngeal cancer,pheochromocytoma, pineal parenchymal tumors, pineoblastoma, pituitarytumor, pleuropulmonary blastoma, primary central nervous systemlymphoma, prostate cancer, rectal cancer, renal cell cancer, salivarygland cancer, sarcoma, Ewing sarcoma, soft tissue sarcoma, squamous cellcarcinoma, Sezary syndrome, skin cancer, Merkel cell carcinoma,testicular cancer, throat cancer, thymoma, thymic carcinoma, thyroidcancer, urethral cancer, endometrial cancer, uterine cancer, uterinesarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia,and Wilms tumor. In one embodiment, the tumor is a glioma. In oneembodiment, the tumor is a tumor other than a glioma.

In one embodiment, the tumor is associated with an amplification or overactivity of an oncogene. In one embodiment, the tumor is associated witha loss or under activity of a tumor suppressor gene. In one embodiment,the tumor suppressor gene is BRCA2. In one embodiment, the tumor isassociated with a loss or under activity of a tumor suppressor gene,other than BRCA2. In one embodiment, the tumor is associated withamplification or over activity of an oncogene and loss or under activityof a tumor suppressor gene. In one embodiment, the tumor is associatedwith a loss or mutation of a gene for a DNA repair enzyme. In oneembodiment, the gene for a DNA repair enzyme is RAD51 or its homolog. Inone embodiment, the tumor is free of a loss or mutation of a gene for aDNA repair enzyme. In one embodiment, the tumor is free of a mutation inthe RAD51 gene or its homolog.

The invention provides methods for increasing uptake and enhancingefficacy of a cell-penetrating polypeptide comprising an anti-DNAantibody or a derivative or variant thereof in targeting tumor or cancercells comprising (a) inducing additional extracellular DNA release at ornear the tumor or cancer cells through the use of a cell-damaging agentor introducing additional extracellular DNA or artificial DNA at or nearthe tumor or cancer cells, (b) administering the cell-penetratingpolypeptide, (c) allowing the cell-penetrating polypeptide to formadditional complexes with the additional extracellular DNA or artificialDNA, and (d) permitting the additional complexes in (c) to contact thetumor or cancer cells, thereby increasing uptake and enhancing efficacyof a cell-penetrating polypeptide comprising an anti-DNA antibody or aderivative or variant thereof in targeting tumor or cancer cells.

Also, the invention provides methods for enhancing the effects ofchemotherapy or radiation therapy by selectively targeting live cells byany of the methods of the invention.

In accordance with the practice of the invention, any of the methods ofthe invention may be an adjunct therapy to a chemotherapy or a radiationtherapy. The chemotherapy or the radiation therapy may be administeredconcurrently or before selectively targeting the live cells by any ofthe methods of the invention.

The invention further provides method for protecting cells(cytoprotection) from a disease or disorder associated with a hydrogenperoxide toxicity or reactive oxygen species (ROS) toxicity by selectivetargeting of live cells at the injury site by any of the methods of theinvention. The disease or disorder may be a brain injury, heart injury,skin injury, or radiation injury and may be an acute injury. Examples ofbrain injury include but are not limited to brain trauma, spinal cordinjury, peripheral nerve injury, or stroke. A heart injury may includebut not limited to a myocardial infarction. Examples of skin injuryinclude but are not limited to wound, burn, or decubitus ulcer. Aradiation injury may include but not limited to burn or poison. Inanother embodiment, the disease or disorder may be acute renal failure,acute organ failure, liver injury, bowel infarction, peripheral vasculardisease, pulmonary failure, or a cancer.

The cell-penetrating polypeptide may be a therapeutic agent. In oneembodiment, the therapeutic agent may be the anti-DNA antibody of theinvention, e.g., 3E10 mAb or its fragment or its variant, free of anyother pharmaceutically active agent. In another embodiment, thecell-penetrating polypeptide may comprise a therapeutic agent linked orcoupled to an anti-DNA antibody of the invention. The therapeutic agentlinked or coupled to an anti-DNA antibody of the invention may be acytotoxic agent, a cytoprotective agent, or another antibody or itsfragment with an intracellular-binding determinant. Theintracellular-binding determinant may be to an oncoprotein, tumorsuppressor gene, a transcription factor, a cell signaling molecule, anuclear receptor, a steroid receptor, a cell signaling molecule, aprotein kinase, a phosphatase, an acetylase, a ligase, a methylase, aprotease, an enzyme, a shuttling protein, a nuclear protein, a nucleolarprotein, transcription components, a soluble protein, a cytoskeletalprotein and a membrane protein.

Additionally, the cell-penetrating polypeptide may be an anti-DNAantibody (e.g., polyclonal, monoclonal, chimeric, bispecific andhumanized antibodies). In one embodiment, the anti-DNA antibody bindsDNA (including single stranded or double stranded DNA). Examples ofsuitable anti-DNA antibodies include, but are not limited to, 3E10antibody, H7 antibody, H9 antibody, H72 antibody, H205 antibody, H317antibody F14-6 antibody, SN22 antibody, SN50 antibody, SN111 antibody,SN112 antibody, SN575 antibody, SN604 antibody, SN608 antibody, F4.1antibody, J20.8 antibody, F14.6 antibody, and 9D7 antibody or aderivative or variant thereof (Vlahakos D, Foster M H, Ucci A A, BarrettK J, Datta S K, and Madaio M P (1992) “Murine monoclonal anti-DNAantibodies penetrate cells, bind to nuclei, and induce glomerularproliferation and proteinuria in vivo.” J. Am. Soc. Nephrol.2(8):1345-54; Ruiz-Argüelles A, Perez-Romano B, Llorente L,Alarcón-Segovia D, and Castellanos J M (1998) “Penetration of anti-DNAantibodies into immature live cells.” J. Autoimmun. 11(5):547-56;Avrameas A, Ternynck T, Nato F, Buttin G, and Avrameas S (1998)“Polyreactive anti-DNA monoclonal antibodies and a derived peptide asvectors for the intracytoplasmic and intranuclear translocation ofmacromolecules” Proc. Natl. Acad. Sci. U.S.A. 95(10):5601-5606; Song YC, Sun G H, Lee T P, Huang J C, Yu C L, Chen C H, Tang S J, and Sun K H(2008) “Arginines in the CDR of anti-dsDNA autoantibodies facilitatecell internalization via electrostatic interactions.” Eur. J. Immunol.38(143178-90). Sequence of CDR for F4.1 antibody, J20.8 antibody andF14.6 antibody may be found in Avrameas A, Ternynck T, Nato F, Buttin G,and Avrameas S (1998) “Polyreactive anti-DNA monoclonal antibodies and aderived peptide as vectors for the intracytoplasmic and intranucleartranslocation of macromolecules” Proc. Natl. Acad. Sci. U.S. A.95(10):5601-5606. Sequence of CDR for 9D7 antibody may be found in SongY C, Sun G H, Lee T P, Huang J C, Yu C L, Chen C H, Tang S J, and Sun KH (2008) “Arginines in the CDR of anti-dsDNA autoantibodies facilitatecell internalization via electrostatic interactions.” Eur. J. Immunol.38(11):3178-90. In one embodiment, the anti-DNA antibody is an isolatedmonoclonal antibody or a derivative or variant thereof. In anembodiment, the isolated monoclonal antibody or a derivative or variantthereof is taken up by live cells in the presence of extracellularnucleic acid, DNA or artificial DNA. In an embodiment, the isolatedmonoclonal antibody or a derivative or variant thereof allows fortargeted uptake of cells with an increased concentration ofextracellular nucleic acid, DNA or artificial DNA. In an embodiment, thecells targeted for uptake of the isolated monoclonal antibody or aderivative or variant are diseased or tumor cells. In an embodiment, theisolated monoclonal antibody or a derivative or variant is targeted to alive cell or any cell within a group of live cells at a site within ananimal or human by targeted delivery of a cytotoxic agent or radiationto induce limited cell death and subsequent release of DNA around ornear the live cell or around, near or in a group of live cells. In oneembodiment, the isolated monoclonal antibody or a derivative or variantis targeted to a live cell or any cell within a group of live cells at asite within an animal or human by targeted delivery of a nucleic acid,DNA or artificial DNA. In one embodiment, such targeted delivery of anucleic acid, DNA or artificial DNA may be achieved by an implant ortransplant of an object, substance, cells or cell-based materialcontaining nucleic acid, DNA or artificial DNA. The implant ortransplant may release nucleic acid, DNA or artificial DNA in a singleburst or over an extended period. In a preferred embodiment, theanti-DNA antibody is an isolated monoclonal antibody 3E10 as produced bya hybridoma having ATCC accession number PTA 2439 or a derivative orvariant thereof. In one embodiment, the variant has a change in theamino acid sequence of a 3E10 scFv, wherein the change in the amino acidsequence does not abolish or prevent cell penetration.

In one embodiment, the cell-penetrating polypeptide is a 3E10 bispecificantibody having an Fv fragment with the cell-penetrating determinantwhich is a 3E10 Fv and a second Fv fragment with an intracellulartarget-binding determinant which is a 3G5 Fv.

In another embodiment, the bispecific antibody comprises an Fv fragmentwith a cell-penetrating determinant of anti-DNA monoclonal antibody 3E10and a second Fv fragment with an intracellular target-bindingdeterminant for MDM2.

In one embodiment, the cell-penetrating polypeptide is a 3E10 bispecificantibody having an Fv fragment with the cell-penetrating determinantwhich is a 3E10 Fv and a second Fv fragment with an intracellulartarget-binding determinant which is a PAb421 Fv.

In another embodiment, the bispecific antibody comprises an Fv fragmentwith a cell-penetrating determinant of anti-DNA monoclonal antibody 3E10and a second Fv fragment with an intracellular target-bindingdeterminant for p53.

Examples of 3E10 bispecific antibodies, e.g. 3E10 scFv and 3G5 scFv aswell 3E10 scFv and PAb421 scFv, are disclosed in U.S. Ser. No.13/844,318, filed Mar. 15, 2013, which is incorporated by referenceherein.

The 3E10 bispecific antibodies of the invention may further comprise oneor more amino acid sequence comprising Ala-Gly-Ile-His (AGIH) at theamino terminus of one or both of the Fv regions. Cell-penetratingpolypeptides may further comprise one or more amino acid sequencecomprising Ala-Gly-Ile-His (AGIH) at the amino terminus.

In another embodiment, the cell-penetrating polypeptide comprises a 3E10Fv attached to a heat shock protein (Hsp). Examples of heat shockproteins include but are not limited to, human Hsp-70 (Hunt and MorimotoPNAS Vol, 82, pp. 64-55-6459, FIGS. 2 and 3); HspA (e.g., HspA1A,HspA1B, HspA1L, HspA2, HspA5, HspA6, HspA7, HspA8, HspA9, HspA12A,HspA12B, HspA13, HspA14); HspH (e.g., HspH1, HspH2, HspH3, and HspH4);Hsp40 (e.g., DnaJA (e.g. DNAJA1, DNAJA2, DNAJA3, and DNAJA4), DnaJB(e.g., DNAJB1, DNAJB2, DNAJB3, DNAJB4, DNAJB5, DNAJB6, DNAJB7, DNAJB8,DNAJB9, DNAJB11, DNAJB12, DNAJB13, and DNAJB14), DnaJC (e.g., DNAJC1,DNAJC2, DNAJC3, DNAJC4, DNAJC5B, DNAJC5G, DNAJC6, DNAJC7, DNAJC8,DNAJC9, DNAJC10, DNAJC11, DNAJC12, DNAJC13, DNAJC14, DNAJC15, DNAJC16,DNAJC17, DNAJC18, DNAJC19, DNAJC20, DNAJC21, DNAJC22, DNAJC23, DNAJC24,DNAJC25, DNAJC26, DNAJC27, DNAJC28, and DNAJC30) and HSPB (HSPB1, HSPB2,HSPB3, HSPB4, HSPB5, HSPB6, HSPB7, HSPB8, HSPB9, HSPB10 and HSPB11)(Kampinga et al., Cell Stress and Chaperones (2009) 14:105-111).

In a further embodiment, the cell-penetrating polypeptide is a fusionprotein comprising a 3E10 Fv joined to a Hsp-70 or portion thereof, andoptionally, the 3E10 Fv comprising an amino acid sequence AGIH at itsamino terminus. Examples of 3E10 fusion proteins are disclosed in U.S.Ser. No. 13/815,829, filed Mar. 15, 2013, which is incorporated byreference herein.

In yet another embodiment, the cell-penetrating polypeptide is a fusionprotein comprising a 3E10 Fv joined to Hsp-27 or portion thereof, andoptionally, the 3E10 Fv comprising an amino acid sequence AGIH at itsamino terminus.

The invention also provides a further embodiment, wherein thecell-penetrating polypeptide is a fusion protein comprising a 3E10 Fvattached or joined to a Hsp-90 or portion thereof, and optionally, the3E10 Fv comprising an amino acid sequence AGIH at its amino terminus.

Additionally, the invention also provides an embodiment, wherein thecell-penetrating polypeptide is a fusion protein comprising a 3E10 Fvattached or joined to GRP78 or portion thereof, and optionally, the 3E10Fv comprising an amino acid sequence AGIH at its amino terminus.

The invention also provides a further embodiment, wherein thecell-penetrating polypeptide is a fusion protein comprising a 3E10 Fvattached or joined to an E3 ubiquitin-protein ligase, a tumorsuppressor-interacting protein, a binding partner of a tumor suppressorprotein, an oncoprotein, or a DNA repair protein or portion thereof, andoptionally, the 3E10 Fv comprising an amino acid sequence AGIH at itsamino terminus. Fusion proteins may be produced by recombinant DNAmethods in which coding sequences isolated from at least two differentsources are assembled in a single nucleic acid molecule so as to allowthe production of a single polypeptide for the fusion protein, alsocalled chimeric protein.

The invention also provides a further embodiment, wherein thecell-penetrating polypeptide is a fusion protein comprising a 3E10 Fvattached or joined to a transcription factor, a transcriptionalrepressor, a transcriptional co-factor, a nuclear receptor, a steroidreceptor, a methylase, an acetylase, a deacetylase, RNA polymerase, akinase, a phosphatase, an intracellular signaling molecule (not a cellsurface signaling molecule), a cell cycle regulatory protein, aprotease, a DNA repair protein, a recombinase, a chromosomal protein, anapoptotic protein, a SUMO ligase, a ubiquitin ligase, a metabolicprotein, an organelle protein, a nuclear protein, a nucleolar protein, amitochondrial protein, a ligand, a ribosomal protein, an enzyme, acytoskeletal protein, a chromosomal protein, a structural protein, aintracellular soluble protein, an intracellular shuttling protein or aregulatory protein or portion thereof, so long as the second determinantfails to recognize any protein that normally resides on the cellsurface. In a further embodiment, the fusion protein comprises an aminoacid sequence AGIH at its amino terminus.

In one embodiment, the cell-penetrating polypeptide is a fusion proteincomprising a 3E10 Fv attached or joined to a nuclear transcriptionfactor, or portion thereof, that is both a tumor suppressor factor andregulatory of T-regulatory cell. In one embodiment, the nucleartranscription factor that is both a tumor suppressor factor andregulatory of T-regulatory cell is Foxp3. In one embodiment, thecell-penetrating polypeptide is a fusion protein comprising a 3E10 Fvattached or joined to Foxp3 or portion thereof. In one embodiment, thecell-penetrating polypeptide is a fusion protein comprising a 3E10 Fvattached or joined to Foxp3 or portion thereof as described in Heinze E,et al., Oncol Lett. 2011 2(4): 665-668.

In one embodiment, the cell-penetrating polypeptide is a fusion proteincomprising a 3E10 Fv attached or joined to a tumor suppressor or portionthereof. In one embodiment, the tumor suppressor factor is a p53 tumorsuppressor protein. In one embodiment, the cell-penetrating polypeptideis a fusion protein comprising a 3E10 Fv attached or joined to p53 tumorsuppressor or portion thereof. In one embodiment, the cell-penetratingpolypeptide is a fusion protein comprising a 3E10 Fv attached or joinedto p53 tumor suppressor or portion thereof as described in Weisbart R H,et al., Cancer Lett. 2003 195(2):211-9.

In yet a further embodiment, the cell-penetrating polypeptide is a 3E10Fv attached to an Hsp-70 or portion thereof, the 3E10 Fv comprising anamino acid sequence AGIH at its amino terminus.

In an additional embodiment, the cell-penetrating polypeptide is a 3E10Fv attached to Hsp-27 or portion thereof, the 3E10 Fv comprising anamino acid sequence AGIH at its amino terminus.

The invention also provides an embodiment, wherein the cell-penetratingpolypeptide is a 3E10 Fv attached to a Hsp-90 or portion thereof, the3E10 Fv comprising an amino acid sequence AGIH at its amino terminus.

In an additional embodiment, the cell-penetrating polypeptide is a 3E10Fv attached to glucose regulated protein 78 kDa (GRP78) or portionthereof, the 3E10 Fv comprising an amino acid sequence AGIH at its aminoterminus.

For example, chimeric antibodies of the invention may be immunoglobulinmolecules that comprise a human and non-human portion. The antigencombining region (variable region) of a chimeric antibody can be derivedfrom a non-human source (e.g. murine) and the constant region of thechimeric antibody which confers biological effector function to theimmunoglobulin can be derived from a human source. The chimeric antibodyshould have the antigen binding specificity of the non-human antibodymolecule and the effector function conferred by the human antibodymolecule.

In general, the procedures used to produce chimeric antibodies caninvolve the following steps:

-   -   a) identifying and cloning the correct gene segment encoding the        antigen binding portion of the antibody molecule; this gene        segment (known as the VDJ, variable, diversity and joining        regions for heavy chains or VJ, variable, joining regions for        light chains or simply as the V or variable region) may be in        either the cDNA or genomic form;    -   b) cloning the gene segments encoding the constant region or        desired part thereof;    -   c) ligating the variable region with the constant region so that        the complete chimeric antibody is encoded in a form that can be        transcribed and translated;    -   d) ligating this construct into a vector containing a selectable        marker and gene control regions such as promoters, enhancers and        poly(A) addition signals;    -   e) amplifying this construct in bacteria;    -   f) introducing this DNA into eukaryotic cells (transfection)        most often mammalian lymphocytes;    -   g) selecting for cells expressing the selectable marker;    -   h) screening for cells expressing the desired chimeric antibody;        and    -   i) testing the antibody for appropriate binding specificity and        effector functions.

A chimeric antibody may include a “humanized” antibody in which one ormore of the complementary-determining region (CDR) from the variableregion of a non-human antibody (such as a mouse monoclonal antibody) maybe used to replace the corresponding CDR in a human antibody, such thatthe resulting chimeric antibody has the framework region of a humanantibody and one or more CDR of a non-human antibody. The chimeric orhumanized antibody may be produced by recombinant DNA methods and may bea whole antibody, an antibody fragment, a bi-specific antibody, a singlechain Fv antibody or combinations thereof.

Antibodies of several distinct antigen binding specificities have beenmanipulated by these protocols to produce chimeric proteins [e.g.anti-TNP: Boulianne et al., Nature 312:643 (1984); and anti-tumorantigens: Sahagan et al., J. Immunol. 137:1066 (1986)]. Likewise,several different effector functions have been achieved by linking newsequences to those encoding the antigen binding region. Some of theseinclude enzymes [Neuberger et al., Nature 312:604 (1984)],immunoglobulin constant regions from another species and constantregions of another immunoglobulin chain [Sharon et al., Nature 309:364(1984); Tan et al., J. Immunol. 135:3565-3567 (1985)]. Additionally,procedures for modifying antibody molecules and for producing chimericantibody molecules using homologous recombination to target genemodification have been described (Fell et al., Proc. Natl. Acad. Sci.USA 86:8507-8511 (1989)).

In accordance with the practice of the invention, the DNA (e.g.,extracellular DNA) may be a single-stranded, double-stranded,triple-stranded, or four-stranded or a combination thereof. Further, theextracellular DNA may comprise a phosphodiester bond, a phosphorothioatebond or a methylphosphonate bond or a combination thereof. For example,the DNA (e.g., extracellular DNA) may comprise a 5′-to-3′ linkage, aninverted 5′-to-5′ linkage or an inverted 3′-to-3′ linkage or acombination thereof. The DNA may be isolated from nature or synthesizedin a laboratory.

In one embodiment, the degradation product of DNA may be bound by theanti-DNA antibody of the invention. DNA degradation products includelower molecular weight DNAs, which may be single-stranded,double-stranded, triple-stranded, or four-stranded or a combinationthereof. Further, DNA degradation products include nucleotides,nucleosides and nucleobases. In one embodiment of the invention, the DNAdegradation product includes a thymine base, thymidine or a thymidinemonophosphate (dTMP). In one embodiment, the thymidine monophosphate maybe a 3′ dTMP, 5′ dTMP or cyclic 3′, 5′ dTMP.

In one embodiment of the invention, the DNA degradation productcomprises a guanine base, deoxyguanosine or a deoxyguanosinemonophosphate (dGMP) or a combination thereof. In one embodiment, thedGMP may be a 3′ dGMP, 5′ dGMP or cyclic 3′, 5′ dGMP.

Further, in one embodiment of the invention, the DNA (e.g.,extracellular DNA) comprises an artificial DNA. For example, theartificial DNA may comprise a DNA mimetic. In one embodiment, the DNAmimetic comprises a pseudopeptide backbone. Merely as examples, thepseudopeptide backbone may comprise any of an ethylglycine, apropylglycine, an ethyl-β-alanine, a propionyl linker, a retro inversolinker, a (S,S)-cyclohexyl linker, a (R,R)-cyclohexyl linker, anL-ornithine, a 2-me-ethyl-glycine, an ethyl-lysine, a L-proline, an-proline, a glycine backbone/ethyl linker, a L-4-trans-amino proline, aL-4-cis-amino proline, a D-4-trans-amino proline, a β-alanine/proline, aglycylglycine/ethyl linker, a glycine/ethyl linker, a proline-glycine, aβ-amino-alanine, E-OPA, Z-OPA, APNA, a serinol-ethyl-methyl linker, aserinol-ethyl-ethyl linker, an α-methyl-serinol-ethyl-ethyl linker, anaminopentan, a hydroxyethyl phosphono glycine, an aminoethyl phosphonoglycine, a lysine, an aminoethyl prolyl or a serinyl methylene orcombination thereof.

Further in accordance with the practice of the invention, thecell-penetrating polypeptide may be whole antibodies or derivativesthereof (e.g., fragments thereof (e.g., Fv, Fab′, F(ab′)₂) orrecombinant proteins including recombinant variable regions ofimmunoglobulin molecules (e.g., scFv, bispecific antibody with scFvfragments)) containing the antigen binding domain and/or one or morecomplement determining regions of these antibodies that penetrate or areinternalized into the cell upon or after binding. These cell-penetratingpolypeptides can be from any source, e.g., rat, dog, cat, pig, horse,mouse or human. It is intended that the term “penetrate” or“internalize” means that the cell-penetrating polypeptide is taken intothe cell. Further, some of the antibodies induce inhibition of cancercell growth. The cell-penetrating polypeptide may be conjugated to atherapeutic agent.

In accordance with the practice of the invention, an antibody fragmentincludes at least a portion of the variable region of the immunoglobulinmolecule that binds to its target, i.e., the antigen binding region.Some of the constant region of the immunoglobulin may also be included.

In an embodiment of the invention, cell penetration is dependent on asalvage pathway. For example, the nucleoside salvage pathway may be apathway mediated by equilibrative nucleoside transporters (ENTs) orSLC29 family of integral membrane proteins. Examples of an equilibrativenucleoside transporter (ENT) or a member of the SLC29 family of integralmembrane proteins is a transporter for purine and pyrimidine nucleosidesand nucleobases or a metabolite thereof. The transporter for purine andpyrimidine nucleosides and nucleobases or a metabolite thereof may be anequilibrative nucleoside transporter ENT2.

In an embodiment of the invention, cell penetration is dependent onbinding to a nucleic acid. In an embodiment of the invention, cellpenetration is dependent on binding to DNA. In an embodiment of theinvention, cell penetration is dependent on binding to an artificialDNA.

In an embodiment of the invention, cell penetration is mediated by ananti-DNA antibody, antibody fragment or derivative or variant whichbinds DNA and a cell surface molecule. In the case of the cell surfacemolecule, binding by the anti-DNA antibody, antibody fragment orderivative or variant to a cell surface molecule may either be direct orindirect binding. An example of a cell surface molecule is a cellsurface polypeptide, carbohydrate, lipid, phospholipid, polycation orpolyanion. An example of a polypeptide cell surface molecule is anequilibrative nucleoside transporter ENT2.

In an embodiment of the invention, cell penetration by the complexformed between the cell-penetrating polypeptide and DNA or itsdegradation product comprises cellular entry by the cell-penetratingpolypeptide. The fate of the bound DNA or its degradation product is notknown.

The term “bind” in the phrase “to bind and penetrate the live cell” (or“to bind and penetrate additional live cells”) by a complex comprising acell-penetrating polypeptide and DNA or its degradation product refersto association of the complex with the live cell at the cell surface.The term “penetrate” in the above phrase refers to penetration of thecell by the cell-penetrating polypeptide; whereas, the fate of the DNAor its degradation product in the complex related to cell-penetration isnot known and remains to be determined.

In one embodiment of the invention, following cell penetration, theanti-DNA antibody, antibody fragment or derivative or variant of theinvention further penetrates or accumulates in the nucleus, e.g., 3E10antibody.

Contemplated in the invention is the use of subcellular localizationsequences linked to the cell-penetrating polypeptide of the invention soas to direct the cell-penetrating polypeptide to a desired cellularcompartment following cell entry. Subcellular localization sequences areshort polypeptides known in the art and may be linked to thecell-penetrating polypeptide by recombinant methods. Subcellularcompartments which may be targeted with subcellular localizationsequences include nucleus, nucleolus, cytoplasm, mitochondria,endoplasmic reticulum, Golgi, and peroxisome.

As used herein recombinant variable regions of immunoglobulin moleculesrefers to variable regions of Ig molecules which are produced bymolecular biological means. Sequences encoding variable domain of theheavy and light chains may be isolated from T-cells, B-cells, leukemiccells, lymphoma cells, or immunoglobulin gene expressing cells, clonedinto expression vector systems, and introduced into a host cell toproduce “recombinant variable regions of immunoglobulin molecules.”Alternatively, the sequences may be recombinantly produced or obtainedfrom genomic DNA. Recombinant antibodies produced in this mannerconsists of an antibody or antibody fragment with the antigen bindingspecificity dependent on the variable region, comprising frameworksequences and CDRs. Such recombinant antibodies may be formed from apolypeptide chain containing a variable region from a light chain and apolypeptide chain containing a variable region from a heavy chain oralternatively both the light chain and heavy chain variable regionscould be found within a polypeptide in which a linker is used to link byrecombinant DNA methods the coding sequences for the two variable chainregions, such as in the case of single chain Fv fragment (scFv).

When recombinant variable regions of immunoglobulin molecules are formedfrom two separate polypeptides, one for the light chain variable regionand other for the heavy chain variable region, the recombinant Igmolecules may be an intact antibody as is normally produced by anorganism from which the coding sequences were isolated or it could be afragment. Antibody fragments could be produced either by recombinant DNAmethods allowing tailored antibodies not dependent on specific proteasecleavage sites or by proteolytic cleavage of the recombinant antibodiessuch as by IdeS, pepsin, or papain to produce Fab, F(ab′) or F(ab′)₂fragments. The “recombinant variable regions of immunoglobulinmolecules” may include the entire constant region or a portion of theconstant region. In addition, the constant region of one antibody may bereplaced by recombinant DNA method with the constant region of adifferent antibody if desired.

Single-chain antibodies or Fv consist of an antibody light chainvariable domain or region (“V_(L)”) and heavy chain variable region(“V_(H)”) connected by a short peptide linker. The peptide linker allowsthe structure to assume a conformation which is capable of binding toantigen].

As used herein, a “conservative amino acid substitution” is thereplacement of one amino acid with another of a similar type such thatthe binding specificity of the antibody is preserved. Amino acids of asimilar type can be classified into several groups in which one aminoacid within a group may be able to substitute for another member of thegroup:

-   (1) non-polar aliphatic amino acids, such as alanine, glycine,    isoleucine, leucine and valine with alanine and glycine more related    to each other and isoleucine, leucine and valine more related to    each other based on size;-   (2) neutral polar amino acids, such as serine, cysteine, threonine,    glutamine and asparagine, and to a lesser extent methionine;-   (3) cyclic amino acid, such as proline;-   (4) aromatic amino acids, such as phenylalanine, tyrosine, and    tryptophan;-   (5) basic amino acids, such as histidine, lysine and arginine;-   (6) acidic amino acids, such as aspartic acid, glutamic acid,    asparagine and glutamine;-   (7) aspartic acid and asparagine;-   (8) glutamic acid and glutamine; and-   (9) alanine, glycine, serine and cysteine

Discussions of conservative amino acid substitution may be found in thepatent literature.

Moreover, the present invention includes nucleic acids with silentmutation or silent mutations. A silent mutation is a mutation in the DNAwhich does not result in a change to the amino acid sequence of aprotein or results in a change to the amino acid sequence of a proteinbut not its functionality. Degeneracy of the genetic code allowsmultiple codons to code for the same amino acid, allowing silentmutations to occur without changing the protein sequence. Such silentmutations are well-known and may be recited readily from publicallyavailable and accepted codon tables. In the case of silent mutations inwhich the amino acid sequence is changed but not the function of theprotein, such silent mutations are generally mutations in which oneamino acid of a certain chemical/physical characteristics is substitutedwith another of a similar type. Such mutations may involve conservativeamino acid substitutions and may be detected through evolutionarychanges but is best determine empirically.

The invention additionally provides a method for detecting an area orzone of cellular turnover. The method comprising (a) contacting cells ata potential area or zone of cellular turnover with a cell-penetratingpolypeptide comprising 3E10 scFv or cell-penetrating determinants of alupus autoantibody or a fragment or variant thereof so that thecell-penetrating polypeptide binds extracellular DNA present at an areaor zone of cellular turnover; (b) permitting cellular uptake of thecell-penetrating polypeptide of (a) at the area or zone of cellularturnover; and (c) detecting the presence of the cell-penetratingpolypeptide inside the cell body, thereby detecting an area or zone ofcellular turnover. In a further embodiment, the method further comprisesidentifying a center of cellular turnover wherein the center marked bypresence of a lysed cell produces a gradient of extracellular DNA suchthat (a) presence of greatest amount of extracellular DNA near thecenter results in greatest uptake of the cell-penetrating polypeptide bylive cells near the center of cellular turnover and (b) presence oflesser amount of extracellular DNA away from the center results in loweruptake of the cell-penetrating polypeptide by live cells away from thecenter of cellular turnover, thereby identifying the center of cellularturnover.

In accordance with the practice of the invention, the cells or tissuemay be from a mammal or derived from a mammal. Examples of mammalsinclude but are not limited to mouse, rat, hamster, cat, dog, rabbit,bovine, pig, sheep, goat, horse, monkey or human.

Cancer immunotherapy using anti-DNA antibodies, alone or combined withextracellular DNA, may follow the teachings generated from variousapproaches which have been successfully employed with respect to othertypes of cancer. For example, one way to apply antitumor monoclonalantibodies clinically is to administer them in unmodified form, usingmonoclonal antibodies of the invention which display antitumor activityand/or internalizing ability and/or in animal models. The anti-tumoractivity of a particular anti-DNA mAb, or combination of anti-DNA mAbs,is preferably evaluated in vivo using a suitable animal model. Xenogeniccancer models, wherein human cancer explants or passaged xenografttissues are introduced into immune compromised animals, such as nude orSCID mice, are particularly appropriate and are known. Examples ofxenograft models of human prostate cancer (capable of recapitulating thedevelopment of primary tumors, micrometastasis, and the formation ofosteoblastic metastases characteristic of late stage disease) are wellknown. The examples herein provide detailed experimental protocols forevaluating the anti-tumor potential of anti-DNA mAb preparations invivo. Other in vivo assays are contemplated, such as those which measureregression of established tumors, interference with the development ofmetastasis, and the like.

The method of the invention contemplates the administration of singleanti-DNA mAbs as well as combinations, or “cocktails”, of differentindividual mAbs such as those recognizing different epitopes. Such mAbcocktails may have certain advantages inasmuch as they contain mAbswhich bind to different epitopes and/or exploit different effectormechanisms or combine directly cytotoxic mAbs with mAbs that rely onimmune effector functionality. Such mAbs in combination may exhibitsynergistic therapeutic effects. In addition, the administration ofanti-DNA mAbs may be combined with other therapeutic agents, includingbut not limited to various chemotherapeutic agents, androgen-blockers,and immune modulators. The anti-DNA mAbs may be administered in their“naked” or unconjugated form, or may have therapeutic agents conjugatedto them.

The anti-DNA monoclonal antibodies used in the practice of the method ofthe invention may be formulated into pharmaceutical compositionscomprising a carrier suitable for the desired delivery method. Suitablecarriers include any material which when combined with the anti-DNA mAbsretains the anti-tumor function of the antibody or cytoprotectivefunction of an antibody conjugate, if cytoprotective effect is desired,and is non-reactive with the subject's immune systems. Examples include,but are not limited to, any of a number of standard pharmaceuticalcarriers such as sterile phosphate buffered saline solutions,bacteriostatic water, and the like (see, generally, Remington'sPharmaceutical Sciences 16^(th) Edition, A. Osal., Ed., 1980).

The anti-DNA antibody formulations may be administered via any routecapable of delivering the antibodies to the tumor site. Potentiallyeffective routes of administration include, but are not limited to,intravenous, intraperitoneal, intramuscular, intratumoral, intradermal,and the like. The preferred route of administration is by intravenousinjection. A preferred formulation for intravenous injection comprisesthe anti-DNA mAbs in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. The anti-DNA mAb preparation may be lyophilized and stored as asterile powder, preferably under vacuum, and then reconstituted inbacteriostatic water containing, for example, benzyl alcoholpreservative, or in sterile water prior to injection.

Treatment may involve the repeated administration of the anti-DNAantibody preparation via an acceptable route of administration such asintravenous injection (IV), at an effective dose. Dosages will dependupon various factors generally appreciated by those of skill in the art,including without limitation the type of cancer and the severity, grade,or stage of the cancer, the binding affinity and half-life of the mAb ormAbs used, the extent of circulating shed DNA, the desired steady-stateantibody concentration level, frequency of treatment, and the influenceof chemotherapeutic agents used in combination with the treatment methodof the invention. Typical daily doses may range from about 0.1 to 100mg/kg. Doses in the range of 10-500 mg mAb per week may be effective andwell tolerated, although even higher weekly doses may be appropriateand/or well tolerated. The principal determining factor in defining theappropriate dose is the amount of a particular antibody necessary to betherapeutically effective in a particular context. Repeatedadministrations may be required in order to achieve tumor inhibition orregression or, alternatively, cytoprotection if cytoprotection isdesired. Initial loading doses may be higher. The initial loading dosemay be administered as an infusion. Periodic maintenance doses may beadministered similarly, provided the initial dose is well tolerated.

In another embodiment, the invention provides methods for selectivelyinhibiting a live cell by reacting any one or a combination of theimmunoconjugates of the invention with the cell in an amount sufficientto inhibit the cell. Such amounts include an amount to kill the cell oran amount sufficient to inhibit cell growth or proliferation. Asdiscussed supra the dose and dosage regimen will depend on the nature ofthe disease or disorder to be treated, its population, the site to whichthe antibodies are to be directed, the characteristics of the particularimmunotoxin, and the patient. For example, the amount of immunoconjugatecan be in the range of 0.1 to 200 mg/kg of patient weight.

In another embodiment, the invention provides a method for increasinguptake and enhancing efficacy of a cell-penetrating polypeptidecomprising an anti-DNA antibody or a fragment or a variant thereof intargeting tumor or cancer cells comprising (a) inducing additionalextracellular DNA release at or near the tumor or cancer cells throughthe use of a cell-damaging agent or introducing additional extracellularDNA or artificial DNA at or near the tumor or cancer cells; (b)administering the cell-penetrating polypeptide; (c) allowing thecell-penetrating polypeptide to form additional complexes with theadditional extracellular DNA or artificial DNA; and (d) permitting theadditional complexes in (c) to contact the tumor or cancer cells,thereby increasing uptake and enhancing efficacy of a cell-penetratingpolypeptide comprising an anti-DNA antibody or a fragment or a variantthereof in targeting tumor or cancer cells.

In another embodiment, the invention provides a method for diagnosing oridentifying a site of cell or tissue injury, an ischemic site withnecrotic or apoptotic cells, a tumor site with necrotic or apoptoticcells, a cancer site with necrotic or apoptotic cells, or a site ofcellular turnover comprising: (a) administering a cell-penetratingpolypeptide comprising cell-penetrating determinants so as tointerrogate one or more sites with the cell-penetrating polypeptide; (b)detecting the presence of the cell-penetrating polypeptide within thenucleus of a live cell; and (c) determining if a cluster of live cellswith the cell-penetrating polypeptide is present at the interrogatedsites, wherein presence of a cluster of live cells with thecell-penetrating polypeptide is indicative of a site of cell or tissueinjury, an ischemic site with necrotic or apoptotic cells, a tumor sitewith necrotic or apoptotic cells, a cancer site with necrotic orapoptotic cells, or a site of cellular turnover, thereby, diagnosing oridentifying a site of cell or tissue injury, an ischemic site withnecrotic or apoptotic cells, a tumor site with necrotic or apoptoticcells, a cancer site with necrotic or apoptotic cells, or a site ofcellular turnover.

In another embodiment, the invention provides a method for increasing orenhancing cytoprotection at an ischemic site comprising: (a)administering DNA to the site in an extracellular space so as to permitincreased targeting of a cell-penetrating polypeptide comprising 3E10scFv or cell-penetrating determinants of a lupus autoantibody or afragment or variant thereof, and a cytoprotective agent; (b)administering the cell-penetrating polypeptide of (a); (c) contactingthe extracellular DNA of (a) or its degradation product at an ischemicsite with a cell-penetrating polypeptide of (a) so that thecell-penetrating polypeptide binds extracellular DNA or its degradationproduct at an ischemic site so as to form a complex; (d) contacting alive cell at risk for dying at an ischemic site with the complex in (c)so as to bind and penetrate the live cell; and (e) permitting additionalcomplexes to form as in (c) and contacting additional live cells withsaid complexes so as to bind and penetrate additional live cells at riskfor dying at an ischemic site;

thereby, delivering additional cytoprotective agent to a cell at risk ofdying and delivering cytoprotective agent to more cells at risk of dyingat an ischemic site, thus, increasing or enhancing cytoprotection at anischemic site.

In one embodiment, the cytoprotective agent is a heat shock protein,stress protein or chaperone protein. The heat shock protein, stressprotein or chaperone protein may be any of Hsp-70, HspA1A, HspA1B,HspA1L, HspA2, HspA5, HspA6, HspA7, HspA8, HspA9, HspA12A, HspA12B,HspA13, HspA14, HspH1, HspH2, HspH3, and HspH4, Hsp40, DNAJA1, DNAJA2,DNAJA3, DNAJA4, DNAJB1, DNAJB2, DNAJB3, DNAJB4, DNAJB5, DNAJB6, DNAJB7,DNAJB8, DNAJB9, DNAJB11, DNAJB12, DNAJB13, DNAJB14, DNAJC1, DNAJC2,DNAJC3, DNAJC4, DNAJC5B, DNAJC5G, DNAJC6, DNAJC7, DNAJC8, DNAJC9,DNAJC10, DNAJC11, DNAJC12, DNAJC13, DNAJC14, DNAJC15, DNAJC16, DNAJC17,DNAJC18, DNAJC19, DNAJC20, DNAJC21, DNAJC22, DNAJC23, DNAJC24, DNAJC25,DNAJC26, DNAJC27, DNAJC28, DNAJC30, HSPB1, HSPB2, HSPB3, HSPB4, HSPB5,HSPB6, HSPB7, HSPB8, HSPB9, HSPB10, HSPB11, hsp90, hsp84, hsp27, hsp20,GRP78, alpha B crystallin, hsp60, hsp100, GRP94, GRP170, AIPL1, FKBP1A,FKBP1B, FKBP2, FKBP3, FKBP5, FKBP6, FKBP7, FKBP8, FKBP9, FKBP9L, FKBP10,FKBP11, FKBP12, FKBP14, FKBP15, FKBP38, FKBP52 and LOC541473.

In one embodiment, an ischemic site is associated with a conditionselected from the group consisting of cardiac ischemia, myocardialinfarction, ischemic colitis, mesenteric ischemia, brain ischemia, acuteischemic stroke, transient ischemic attack, vascular dementia, stroke,acute limb ischemia, cyanosis, gangrene, an embolism, a thrombosis, anatherosclerosis artery, a trauma, venous outflow obstruction, acutearterial ischemia, an aneurysm, mitral valve disease, chronic atrialfibrillation, cardiomyopathies, an occlusion, pulmonary embolus, acutearterial occlusion, peripheral arterial disease, a thromboembolism, acompression, a shearing, a laceration, arterial dissection, iatrogenicarterial injury, thoracic outlet syndrome, atherosclerosis,hypoglycemia, tachycardia, hypotension, septic shock, heart failure,superior mesenteric artery syndrome, sickle cell disease, inducedg-force, frostbite, improper cold compression therapy, tourniquetapplication, increased glutamate receptor stimulation, arteriovenousmalformation, peripheral artery occlusive disease, rupture ofsignificant blood vessel, anemia, cardiac arrhythmia, cardiorespiratoryarrest, subarachnoid hemorrhage, intracerebral hemorrhage, cerebralinfarction, focal brain ischemia, global brain ischemia, pulmonaryinfarction, lung infarction, splenic infarction, limb infarction, deepvein thrombosis, phlebitis, skeletal muscle infarction, diabetesmellitus, avascular necrosis, testicular torsion, testicular infarction,central retinal artery infarction, sepsis, antiphospholipid syndrome,giant-cell arteritis, hemia, volvulus, hepatic ischemia, dehydration andinfection.

Compositions

The invention provides a pharmaceutical composition comprising ananti-DNA antibody, fragment or derivative or variant thereof and,optionally, a suitable carrier. The invention also provides acomposition or pharmaceutical composition comprising a cell-penetratingpolypeptide which comprises cell-penetrating determinants andextracellular DNA or artificial DNA. The invention also provides acomposition or pharmaceutical composition comprising a cell-penetratingpolypeptide which comprises cell-penetrating determinants andextracellular DNA or artificial DNA, and optionally, a suitable carrier.The antibody or fragment or derivative or variant thereof may beconjugated or linked to a therapeutic drug or a cytotoxic agent. Theantibody or fragment or derivative or variant thereof may be conjugatedor linked to a hapten, an epitope tag or an imaging agent. The antibodyor fragment or derivative thereof may be conjugated or linked to acytoprotective agent. The antibody or fragment or derivative thereof maybe conjugated or linked to a chemical compound, a peptide or a protein.

Suitable carriers for pharmaceutical compositions include any materialwhich when combined with the nucleic acid or other molecule of theinvention retains the molecule's activity and is non-reactive with thesubject's immune systems. Examples include, but are not limited to, anyof the standard pharmaceutical carriers such as a phosphate bufferedsaline solution, water, emulsions such as oil/water emulsion, andvarious types of wetting agents. Other carriers may also include sterilesolutions, tablets including coated tablets and capsules. Typically suchcarriers contain excipients such as starch, milk, sugar, certain typesof clay, gelatin, stearic acid or salts thereof, magnesium or calciumstearate, talc, vegetable fats or oils, gums, glycols, or other knownexcipients. Such carriers may also include flavor and color additives orother ingredients. Compositions comprising such carriers are formulatedby well-known conventional methods. Such compositions may also beformulated within various lipid compositions, such as, for example,liposomes as well as in various polymeric compositions, such as polymermicrospheres.

Anti-DNA Antibodies

The invention provides anti-DNA antibodies for use in the methods andcompositions of the invention. In one embodiment, the anti-DNAantibodies which may be used in the invention includes any of H7Antibody, H9 Antibody, H72 Antibody, H205 Antibody, H317 Antibody F14-6Antibody, SN22 Antibody, SN50 Antibody, SN111 Antibody, SN112 Antibody,SN575 Antibody, SN604 Antibody, Sn608 Antibody, F4.1 Antibody, J20.8Antibody, F14.6 Antibody, and 9D7 antibody or a derivative or variantthereof (Vlahakos D, Foster Mh, Ucci Aa, Barrett Kj, Datta Sk, AndMadaio Mp (1992) “Murine Monoclonal Anti-Dna Antibodies Penetrate Cells,Bind To Nuclei, And Induce Glomerular Proliferation And Proteinuria InVivo.” J. Am. Soc. Nephrol. 2(8):1345-54; Ruíz-ArgUelles A, Pérez-RomanoB, Llorente L, Alarcón-Segovia D, And Castellanos Jm (1998) “PenetrationOf Anti-DNA Antibodies Into Immature Live Cells.” J. Autoimmun.11(5):547-56; Avrameas A, Ternynck T, Nato F, Buttin G, And Avrameas S(1998) “Polyreactive Anti-Dna Monoclonal Antibodies And A DerivedPeptide As Vectors For The Intracytoplasmic And IntranuclearTranslocation Of Macromolecules” Proc. Natl. Acad. Sci. U.S.A.95(10):5601-5606; Song Yc, Sun Gh, Lee Tp, Huang Jc, Yu Cl, Chen Ch,Tang Sj, And Sun Kh (2008) “Arginines In The CDR Of Anti-dsDNAAutoantibodies Facilitate Cell Internalization Via ElectrostaticInteractions.” Eur. J. Immunol. 38(11):3178-90). Sequence Of CDR ForF4.1 Antibody, J20.8 Antibody And F14.6 Antibody May Be Found InAvrameas A, Ternynck T, Nato F, Buttin G, And Avrameas S (1998)“Polyreactive Anti-DNA Monoclonal Antibodies And A Derived Peptide AsVectors For The Intracytoplasmic And Intranuclear Translocation OfMacromolecules” Proc. Natl. Acad. Sci. U.S. A. 95(10):5601-5606.Sequence Of CDR For 9d7 Antibody May Be Found In Song Yc, Sun Gh, LeeTp, Huang Jc, Yu Cl, Chen Ch, Tang Sj, And Sun Kh (2008) “Arginines InThe CDR Of Anti-DsDNA Autoantibodies Facilitate Cell Internalization ViaElectrostatic Interactions.” Eur. J. Immunol. 38(11):3178-90. In OneEmbodiment, The Anti-DNA Antibodies Which May Also Be Used In TheInvention Includes 5c5 Monoclonal Antibody, 5c6 Monoclonal ClonalAntibody And 4h2 Monoclonal Antibody (Weisbart Rh, Et Al., J. Immunol.1990 144(7): 2653-2658; Zack Dj, Et Al., J. Immunol. 1995 154(4):1987-1994; Weidle Uh, Et Al., Cancer Genomics Proteomics 2013 10:239-250; Weisbart Rh, Et Al., Sci. Rep. 2015 5: 12022; Noble Pw, Et Al.,Sci. Rep. 2014 4:5958; Colburn Kk, Et Al., J. Rheumatol. 200330(5):993-7).

3E10 Antibodies

The invention further provides an example of an anti-DNA antibody whichis a 3E10 antibody (e.g., polyclonal, monoclonal, chimeric, andhumanized antibodies) for use in the methods and compositions of theinvention. Anti-3E10 antibodies that are particularly contemplatedinclude monoclonal ntibodies as well as fragments thereof (e.g.,recombinant proteins, such as scFv) containing the antigen bindingdomain and/or one or more complement determining regions of theseantibodies. 3E10 Mab is a murine monoclonal antibody and its nucleicacid sequence and amino acid sequence provided in: FIGS. 3 and 4 Of ZackDj, Et Al., J. Immunol. 1995 154(4): 1987-1994; FIGS. 3 And 4 Of UsPatent Application Publication No.: US 2008/0292618 A1; FIGS. 1 and 2 ofPCT International Publication No.: WO 2010/138769 A1, published 2 Dec.2010; GenBank Accession Numbers: L16982 for mAb 3E10 Vh chain and L34051for mAb 3E10 vic light chain. location of the complement-determiningregions (e.g., CDR1, CDR2 and CDR3) Along With The Framework Regions(i.e., FR1, FR2, FR3, and FR4) of the 3E10 variable heavy chain andlight chain domains are provided in FIGS. 3 and 4 of Zack Dj, Et Al., J.Immunol. 1995 154(4): 1987-1994; FIGS. 3 and 4 of US Patent ApplicationPublication No.: US 2008/0292618 A1; FIGS. 1 and 2 of PCT InternationalPublication No. WO 2010/138769 A1, published 2 Dec. 2010. In a preferredembodiment, the anti-3E10 antibody or its fragment is a variant, such asthe D31N 3E10 variant in which amino acid residue 31 of 3E10 variableheavy chain is mutated from an aspartic acid (D) to an asparagine (N).This D31N variant of 3E10 antibody has increased binding to ssDNA AnddsDNA (Zack Dj, Et Al., J. Immunol. 1995 154(4): 1987-1994) and enhancedcell and nuclear penetration (Zack Dj, Et Al., J. Immunol. 1996 157(5):2082-2088; Weisbart Wh, et al., J. Autoimmunity 1998 11(5): 539-546). Inan embodiment of the invention, the 3E10 antibody or its fragment orvariant is a derivative. In another embodiment, a derivative may be inthe form of an immunoconjugate. Such immunoconjugates are discussedsupra.

Humanized Antibodies

The present invention encompasses humanized antibodies. Various methodsfor humanizing non-human antibodies are known in the art.

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to one method, humanized antibodies areprepared by a process of analysis of the parental sequences and variousconceptual humanized products using three-dimensional models of theparental and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart.

Human Antibodies

Human antibodies of the invention can be constructed by combining Fvclone variable domain sequences selected from human-derived phagedisplay libraries with known human constant domain sequences.Alternatively, human monoclonal antibodies of the invention can be madeby the hybridoma method. Human myeloma and mouse-human heteromyelomacell lines for the production of human monoclonal antibodies are wellknown in the art. Gene shuffling can also be used to derive humanantibodies from non-human, where the human antibody has similaraffinities and specificities to the starting non-human antibody using amethod called epitope imprinting.

Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. Bispecific antibodies may be obtained from intact antibodiesor antibody fragments. Methods for making bispecific antibodies areknown in the art and described herein. Antibodies with more than twovalencies are contemplated. For example, trispecific antibodies can beprepared.

Antibody Variants

In some embodiments, amino acid sequence modifications of the antibodiesdescribed herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of the antibody are prepared byintroducing appropriate nucleotide changes into the antibody nucleicacid, or by peptide synthesis. Such modifications include, for example,deletions from, and/or insertions into and/or substitutions of, residueswithin the amino acid sequences of the antibody. Any combination ofdeletion, insertion, and substitution is made to arrive at the finalconstruct, provided that the final construct possesses the desiredcharacteristics. The amino acid alterations may be introduced in thesubject antibody amino acid sequence at the time that sequence is made.Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Another type of variant is an amino acidsubstitution variant. These variants have at least one amino acidresidue in the antibody molecule replaced by a different residue. Thesites of greatest interest for substitutional mutagenesis include thehypervariable regions, but FR alterations are also contemplated. Forexample, in one preferred embodiment of the 3E10 antibody, amino acidresidue 31 of 3E10 variable heavy chain is mutated from an aspartic acid(D) to an asparagine (N) to produce the D31N variant of 3E10 antibody.This D31N variant of 3E10 antibody has increased binding to ssDNA anddsDNA (Zack D J, et al., J. Immunol. 1995 154(4): 1987-1994) andenhanced cell and nuclear penetration (Zack D J, et al., J. Immunol.1996 157(5): 2082-2088; Weisbart W H, et al., J. Autoimmunity 11(5):539-546).

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

Antibody Derivatives

The antibodies of the present invention can be further modified tocontain additional proteinaceous or nonproteinaceous moieties that areknown in the art. Proteinaceous or nonproteinaceous moieties include,but are not limited to, chemical functional groups such as hydrophiliclinkers (e.g. polyethylene glycol), crosslinking agents, metalchelators, epitope tags, peptides such as AGIH tetrapeptide sequence,cytotoxic agents, enzymes, cytoprotective agents, a second antibody (orfragment or variant thereof), imaging agents or detectable markers.Suitable examples of the imaging agent or detectable marker include aradioisotope, a fluorophore, a fluorescent quencher, an enzyme, aluminescent compound, a chemiluminescent compound, a bioluminescentcompound, a photon emitter, a heavy metal, a ferromagnetic agent, acontrast agent, a metal chelator, and an epitope.

For example, derivatives of anti-DNA antibodies of the invention (suchas 3E10 antibody) may be a fusion protein comprising thecell-penetrating determinants of the anti-DNA antibody (such as 3E10antibody) and a second biologically active desired functional protein orpeptide.

Nucleic Acid Molecules

In an embodiment, the invention provides a nucleic acid moleculeencoding the anti-DNA antibodies in the compositions of the invention.The nucleic acid molecule may encode the anti-DNA antibodies in thecompositions of the invention.

The nucleic acids of the invention may comprise nucleotide sequences andpolypeptides encoding amino acid sequences which are at least about 70%identical, preferably at least about 80% identical, more preferably atleast about 90% identical and most preferably at least about 95%identical (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to the referencenucleotide and amino acid sequences of the present invention (i.e., seeexample herein) when the comparison is performed by a BLAST algorithmwherein the parameters of the algorithm are selected to give the largestmatch between the respective sequences over the entire length of therespective reference sequences. Polypeptides comprising amino acidsequences which are at least about 70% similar, preferably at leastabout 80% similar, more preferably at least about 90% similar and mostpreferably at least about 95% similar (e.g., 95%, 96%, 97%, 98%, 99%,100%) to the reference amino acid sequences of the present inventionwhen the comparison is performed with a BLAST algorithm wherein theparameters of the algorithm are selected to give the largest matchbetween the respective sequences over the entire length of therespective reference sequences, are also included in the presentinvention.

The nucleic acid molecule may be a DNA molecule (e.g., cDNA) encodingthe bispecific composition of the invention. For example, the inventionprovides for a DNA construct comprising a vector that expresses thebispecific composition of the invention.

Additionally, the invention provides a vector which comprises thenucleic acid molecule of the invention. The host vector system comprisesthe vector of the invention in a suitable host cell. Examples ofsuitable host cells include but are not limited to bacterial cell andeukaryotic cells.

Vectors, Host Cells and Recombinant Methods

For recombinant production of an antibody of the invention, the nucleicacid encoding it is isolated and inserted into a replicable vector forfurther cloning (amplification of the DNA) or for expression. DNAencoding the antibody is readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors are available. The choice ofvector depends in part on the host cell to be used. Generally, preferredhost cells are of either prokaryotic or eukaryotic (generally mammalian)origin. It will be appreciated that constant regions of any isotype canbe used for this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species.

Immunoconjugates

The invention also provides immunoconjugates (interchangeably termed“antibody-drug conjugates” or “ADC”), comprising any of the anti-DNAantibodies described herein conjugated to a cytotoxic agent such as achemotherapeutic agent, a drug, a growth inhibitory agent, a toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate). Immunoconjugates may also comprise any of the anti-DNAantibodies described herein conjugated to a cytoprotective agent such asa heat shock protein, stress protein or chaperone protein.

Chemotherapeutic agents useful in the generation of immunoconjugates aredescribed herein. Conjugates of an antibody and one or more smallmolecule toxins, such as a calicheamicin, maytansinoids, dolastatins,aurostatins, a trichothecene, and CC1065, and the derivatives of thesetoxins that have toxin activity, are also contemplated herein. Otherantitumor agents that can be conjugated to the antibodies of theinvention include BCNU, streptozoicin, vincristine and 5-fluorouracil.Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, saponariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent may be made, e.g., by recombinant techniques or peptide synthesis.The length of DNA may comprise respective regions encoding the twoportions of the conjugate either adjacent one another or separated by aregion encoding a linker peptide which does not destroy the desiredproperties of the conjugate.

In an embodiment, heat shock protein, stress protein or chaperoneprotein include any of Hsp-70, HspA1A, HspA1B, HspA1L, HspA2, HspA5,HspA6, HspA7, HspA8, HspA9, HspA12A, HspA12B, HspA13, HspA14, HspH1,HspH2, HspH3, and HspH4, Hsp40, DNAJA1, DNAJA2, DNAJA3, DNAJA4, DNAJB1,DNAJB2, DNAJB3, DNAJB4, DNAJB5, DNAJB6, DNAJB7, DNAJB8, DNAJB9, DNAJB11,DNAJB12, DNAJB13, DNAJB14, DNAJC1, DNAJC2, DNAJC3, DNAJC4, DNAJC5B,DNAJC5G, DNAJC6, DNAJC7, DNAJC8, DNAJC9, DNAJC10, DNAJC11, DNAJC12,DNAJC13, DNAJC14, DNAJC15, DNAJC16, DNAJC17, DNAJC18, DNAJC19, DNAJC20,DNAJC21, DNAJC22, DNAJC23, DNAJC24, DNAJC25, DNAJC26, DNAJC27, DNAJC28,DNAJC30, HSPB1, HSPB2, HSPB3, HSPB4, HSPB5, HSPB6, HSPB7, HSPB8, HSPB9,HSPB10, HSPB11, hsp90, hsp84, hsp27, hsp20, GRP78, alpha B crystallin,hsp60, hsp100, GRP94, GRP170, AIPL1, FKBP1A, FKBP1B, FKBP2, FKBP3,FKBP5, FKBP6, FKBP7, FKBP8, FKBP9, FKBP9L, FKBP10, FKBP11, FKBP12,FKBP14, FKBP15, FKBP38, FKBP52 and L00541473.

Immunoconjugate of any anti-DNA antibody described herein and acytoprotective agent may be produced by any coupling method known in theart, including chemical crosslinking and recombinant methods.

The antibody or fragment thereof of the invention may be labeled with adetectable marker or conjugated to a second molecule, such as atherapeutic agent (e.g., a cytotoxic agent or cytoprotective agent)thereby resulting in an immunoconjugate. For example, the therapeuticagent includes, but is not limited to, an anti-tumor drug, a toxin, aradioactive agent, a cytokine, a second antibody, an enzyme, asubstrate, a heat shock protein, a stress protein or a chaperoneprotein. Further, the invention provides an embodiment wherein theantibody of the invention is linked to an enzyme that converts a prodruginto a cytotoxic drug. The invention provides an embodiment wherein theantibody of the invention is linked to an enzyme that participates in adenatured protein response or protein refold. The invention provides anembodiment wherein the antibody of the invention is linked to substratethat participates in a detoxification process or an enzyme thatdetoxifies, such as glutathione S-transferase, cytochrome P450 oxidase,UDP-glucuronosyltransferase or alcohol dehydrogenase.

Examples of cytotoxic agents include, but are not limited to ricin,ricin A-chain, doxorubicin, daunorubicin, taxol, ethidium bromide,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine,dihydroxy anthracenedione, actinomycin D, diphtheria toxin, Pseudomonasexotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain,alpha-sarcin, gelonin, mitogellin, restrictocin, phenomycin, enomycin,curcin, crotin, calicheamicin, saponaria officinalis inhibitor,maytansinoids, and glucocorticoid and other chemotherapeutic agents, aswell as radioisotopes such as ²¹²Bi, ¹³¹, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.Suitable detectable markers include, but are not limited to, aradioisotope, a fluorescent compound, a bioluminescent compound,chemiluminescent compound, a metal chelator or an enzyme. Antibodies mayalso be conjugated to an anti-cancer pro-drug activating enzyme capableof converting the pro-drug to its active form.

Additionally, the recombinant protein of the invention comprising theantigen-binding region of any of the monoclonal antibodies of theinvention can be used to treat cancer. In such a situation, theantigen-binding region of the recombinant protein is joined to at leasta functionally active portion of a second protein having therapeuticactivity. The second protein can include, but is not limited to, anenzyme, lymphokine, oncostatin, toxin or a second antibody directed to adifferent antigen. Suitable toxins include those described above.

Techniques for conjugating (e.g., by chemical means) or joining (byrecombinant means) therapeutic agents to antibodies are well known (see,e.g., Amon et al., “Monoclonal Antibodies For Immunotargeting Of DrugsIn Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy,Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstromet al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2ndEd.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987);Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: AReview”, in Monoclonal Antibodies '84: Biological And ClinicalApplications, Pinchera et al. (eds.), pp. 475-506 (1985); and Thorpe etal., “The Preparation And Cytotoxic Properties Of Antibody-ToxinConjugates”, Immunol. Rev., 62:119-58 (1982)).

Pharmaceutical Formulations

Therapeutic formulations comprising an antibody of the invention areprepared for storage by mixing the antibody having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington: The Science and Practice of Pharmacy 20thedition (2000)), in the form of aqueous solutions, lyophilized or otherdried formulations. Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, histidine and other organicacids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended. In one embodiment, the formulationcontains an anti-DNA antibody or fragments, derivatives or variantsthereof, as described herein and DNA (extracellular DNA) as the onlytherapeutic agents in the formulation.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington: The Science and Practice of Pharmacy 20th edition (2000).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes. When appropriate, chemical or radiation sterilization methodmay be used.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the immunoglobulin of the invention,which matrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and γ-ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated immunoglobulins remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

In one embodiment, the sustained-release formulation comprises acell-penetrating polypeptide and a carrier. In another embodiment, thesustained-release formulation comprises a cell-penetrating polypeptideand a preservative. In another embodiment, the sustained-releaseformulation comprises DNA and/or its degradation product(s) and acarrier. In another embodiment, the sustained-release formulationcomprises DNA and/or its degradation product(s) and a preservative. Inone embodiment, the sustained-release formulation comprises acell-penetrating polypeptide and DNA and/or its degradation products. Inone embodiment, the sustained-release formulation comprises acell-penetrating polypeptide and DNA and/or its degradation products anda carrier. In one embodiment, the sustained-release formulationcomprises a cell-penetrating polypeptide and DNA and/or its degradationproducts. In one embodiment, the sustained-release formulation comprisesa cell-penetrating polypeptide and DNA and/or its degradation productsand a preservative.

The formulation may be an immediate-release formulation. In oneembodiment, the immediate-release formulation comprises acell-penetrating polypeptide and a carrier. In another embodiment, theimmediate-release formulation comprises a cell-penetrating polypeptideand a preservative. In another embodiment, the immediate-releaseformulation comprises DNA and/or its degradation product(s) and acarrier. In another embodiment, the immediate-release formulationcomprises DNA and/or its degradation product(s) and a preservative. Inone embodiment, the immediate-release formulation comprises acell-penetrating polypeptide and DNA and/or its degradation products. Inone embodiment, the immediate-release formulation comprises acell-penetrating polypeptide and DNA and/or its degradation products anda carrier. In one embodiment, the immediate-release formulationcomprises a cell-penetrating polypeptide and DNA and/or its degradationproducts. In one embodiment, the immediate-release formulation comprisesa cell-penetrating polypeptide and DNA and/or its degradation productsand a preservative.

In a further embodiment, the formation is a combination of animmediate-release formulation and a sustained-release formulation.

Use of immunologically reactive fragments, such as the Fab, Fab′, orF(ab)₂ fragments or scFv is often preferable, especially in atherapeutic context, as these fragments are generally less immunogenicthan the whole immunoglobulin. Further, bi-specific antibodies specificfor two or more epitopes may be generated using methods generally knownin the art. Further, antibody effector functions may be modified so asto enhance the therapeutic effect of 3E10 antibodies on cancers.Homodimeric antibodies may also be generated by cross-linking techniquesknown in the art (e.g., Wolff et al., Cancer Res. 53: 2560-2565). Theinvention also provides pharmaceutical compositions having themonoclonal antibodies or anti-idiotypic monoclonal antibodies of theinvention, such as anti-idiotypic mAb 1C7 directed against anti-DNAantibody including 3E10 mAb (Weisbart R H et al., J. Immunol. 1990144(7): 2653-2658). Such anti-idiotypic antibodies may be useful inneutralizing activity of an anti-DNA antibody or 3E10 antibody or afragment, variant or derivative thereof.

The following examples are intended to illustrate the present invention,not to limit the scope of the invention in any way.

Example 1 Methods and Materials

Production and Purification of 3E10 scFv.

3E10 scFv used in these studies was previously modified by a D31Nmutation in CDR1 of the variable region of the heavy chain that resultsin a 50-fold increase in DNA-binding affinity and efficiency of nuclearpenetration. 3E10 scFv was produced in P. pastoris and purified asdescribed previously (10).

Cell Lines and Tissue Culture.

The GM02605 human fibroblast cell line (Coriell Biorepository, Camden,N.J.) was selected for these studies because it grows to confluence in96-well tissue culture plates with remarkably high viability (>99%viability maintained over several days of growth as determined bypropidium iodide exclusion assay). Cells were grown in MEM with 10% FCSand washed with MEM without serum before incubation with 10 μM 3E10 scFvfor one hour. Nuclear penetration by 3E10 scFv was then examined byanti-Myc immunostaining as previously described (10).

Cell Lysate.

COS-7 cell lysate was prepared by subjecting cells to multiplefreeze-thaw cycles in liquid nitrogen. Cell debris was removed bycentrifugation. DNA-depleted COS-7 cell lysate was prepared by passingthe lysate through a Centricon cellulose filter with a molecular weightcut off of 10,000 Da.

DNA.

Purified calf thymus DNA sheared to an average length of 2000 bp waspurchased from Invitrogen (Ultrapure, Invitrogen, Carlsbad, Calif.).

Human Glioma Xenografts.

U87 human glioma subcutaneous xenografts were generated in nude mice.When tumors reached size of 100 mm³ mice were treated withintraperitoneal injection (IP) of control PBS buffer or 0.8 mg 3E10 scFvin PBS. Mice were sacrificed 4 or 24 hours after treatment, and tumorsand selected normal tissues were fixed in formalin and embedded inparaffin. Tissues were then surveyed for nuclear penetration by 3E10scFv by immunohistochemistry (IHC) as previously described (11).Briefly, sections were incubated with 9E10 anti-Myc (Invitrogen) primaryantibody directed at the C-terminal Myc tag in 3E10 scFv before probingwith secondary antibody and visualizing with 3,3′-diaminobenzidine andcounterstaining with hematoxylin.

Results

A Factor Released by Dead Cells Appears to Enhance Nuclear Penetrationby 3E10 scFv.

We sought to test the efficiency of nuclear penetration by 3E10 scFvinto cells in the absence of extracellular DNA, and for these studiesselected the GM02605 human fibroblast cell line because it maintains ahigh degree of viability (>99%) with minimal cell death even whilemaintained in culture for several days. With low rates of turnover theconfounding effects of DNA released by dead cells are minimized. TheGM02605 cells were washed with serum free media and then treated with 10μM 3E10 scFv for one hour, after which cells were fixed andimmunostained for presence of the fragment. Remarkably, 3E10 scFv didnot penetrate into most cells. Instead, 3E10 scFv was detected only inthe nuclei of cells centered around what appeared to be a dead cell,with a gradient effect observed with diminishing amounts of intranuclearantibody detected with increasing distance from the central dead cell. Arepresentative image demonstrating this effect is shown in FIG. 1. Thisobservation was consistent with a factor released locally by dying cellsenhancing nuclear penetration of 3E10 scFv into surrounding cells.

Addition of Cell Lysate Promotes Homogenous Nuclear Uptake of 3E10 scFv.

To test the hypothesis that a factor released by dead cells enhancesnuclear uptake of 3E10 scFv we next compared the efficiency of nuclearpenetration of the fragment into the GM02605 fibroblasts in the presenceor absence of a cell lysate. As shown in FIG. 2, in the absence of thecell lysate minimal nuclear uptake of 3E10 scFv was observed. However,the addition of cell lysate facilitated nuclear penetration by 3E10 scFvinto ˜100% of the cells. These results further support the hypothesisthat a factor released by dead cells contained in cell lysate promotesnuclear uptake of 3E10 scFv.

DNA-Depleted Cell Lysate does not Enhance Nuclear Uptake of 3E10 scFv.

Based on our previous observations that 3E10 scFv binds DNA and isunable to penetrate cells that are deficient in the ENT2 nucleosidetransporter, we hypothesized that DNA is the critical factor in celllysate that promotes nuclear uptake of 3E10 scFv. To test this, theGM06205 fibroblasts were treated with 10 μM 3E10 scFv in the presence ofcell lysate that had been filtered to remove DNA content. In contrast tothe complete cell lysate, the DNA-depleted lysate did not enhancenuclear uptake of 3E10 scFv (FIG. 2), which strongly supports thehypothesis that DNA is the relevant factor contributing to nuclearpenetration by the fragment.

Addition of Purified DNA Promotes Homogeneous Nuclear Uptake of 3E10scFv.

To confirm that extracellular DNA enhances nuclear penetration by 3E10scFv, we next treated the GM02605 fibroblasts with 3E10 scFv in thepresence of purified DNA. As shown in FIG. 2, addition of purified DNAto the media significantly enhanced the efficiency of penetration by3E10 scFv into cell nuclei. Taken together, these data indicate thatnuclear penetration by 3E10 scFv is enhanced by the presence ofextracellular DNA.

3E10 scFv Targets Tumor Cells In Vivo.

Based on the observation that 3E10 scFv penetrates cell nuclei mostefficiently in the presence of extracellular DNA, we hypothesized thatwhen administered in vivo the fragment would accumulate most efficientlyinto tissues in which there would be expected to be a higherconcentration of extracellular DNA due to high cellular turnover, suchas is associated with tumors. To test this, subcutaneous U87 humanglioma xenografts were generated in immunodeficient mice, and oncetumors grew to size of 100 mm³ mice were treated with intraperitonealinjection of control buffer or 3E10 scFv. Mice were then sacrificed 4 or24 hours after treatment, and tumors and select normal tissues wereimmunostained for the presence of 3E10 scFv. As shown in FIG. 3A, fourhours after treatment 3E10 scFv was detected in the nuclei of the U87xenograft cells, but was not detected in tissues of major organsincluding heart, kidney, skeletal muscle, and liver. 3E10 scFv was alsodetected in the tumors 24 hours after treatment, demonstrating thestability of the uptake into tumor nuclei (FIG. 3B). These results areconsistent with enhanced uptake of 3E10 scFv into sites of high cellturnover where DNA is released from dying cells.

Discussion

3E10 scFv has been explored as a therapeutic intracellular transportsystem and has successfully mediated delivery of p53, Hsp70, and theanti-MDM2 antibody 3G5 to target tissues in vivo. In addition, 3E10 byitself has been shown to sensitize tumors in vivo to DNA-damaging agentsincluding ionizing radiation and doxorubicin. Importantly, 3E10 and 3E10scFv have never been found to be significantly toxic to any normaltissues in any of these previous in vivo studies. The present study wascarried out to further evaluate preliminary evidence that 3E10 scFvpreferentially targets ischemic tissue or areas of high cell turnoverrates such as malignant tissue. For example, we previously found thatthe Fv-Hsp70 fusion protein protected rats from reperfusion injury ofischemic brain even when administered 3 hours after ligation of themiddle cerebral artery, and in this study 3E10 scFv was shown tolocalize in ischemic but not normoxic brain (9). These findings wereconsistent with a previous study that showed targeting of ananti-histone antibody to an area of cell death in vivo (12). We alsoobserved that the 3E10-3G5 bispecific antibody yielded a profoundsuppression of MDM2-addicted tumors in vivo but showed a remarkableabsence of systemic toxicity (13), suggesting a preferentiallocalization of the agent to tumor cells.

In the present study we have now shown that penetration of 3E10 scFvinto live cells in vitro requires the presence of extracellular DNA. Ofnote, our findings are consistent with a previous report thatdemonstrated the requirement of extracellular DNA for penetration of ananother anti-DNA antibody into living T-cells, however only 10% of cellsinternalized antibody (14). Moreover, when 3E10 scFv was administeredsystemically it was observed to preferentially localize into U87 cancercells implanted subcutaneously, consistent with the fact that rapidlygrowing cancers are ischemic and have a high level of cell turnover andtherefore the local environment should be enriched for extracellularDNA. The selective targeting of 3E10 scFv to such tissues may thereforeexplain in part the remarkable lack of off-target toxicity of 3E10 aloneand of 3E10 scFv-p53 and 3E10-3G5 administered systemically in ourprevious studies.

We previously showed that 3E10 scFv penetrates living cells through theENT2 nucleoside salvage pathway, and our results here suggest that 3E10scFv bound to DNA may be processed by membrane nucleases andphosphatases into fragments that are accessible to this pathway. Furtherstudies are required to characterize these membrane-related events, butthe primary significance of our study is the demonstration that 3E10scFv has targeting specificity in vivo to areas of tissue ischemia andhigh cell turnover. This finding further establishes the potential touse 3E10 scFv in a variety of clinical applications that includeprotecting organs from reperfusion injury and cytotoxic applications forcancer therapy. In addition, recognition of the requirement forextracellular DNA for nuclear penetration by 3E10 scFv suggests thatcombinations of 3E10 scFv with targeted approaches that selectivelyincrease cell turnover in tumors, such as locally applied radiotherapy,might facilitate the subsequent penetration of 3E10 scFv into all cellscomprising a tumor mass due to enhanced release of DNA by dying cells.

REFERENCES

-   1. Alarcon-Segovia, D. (2001) Antinuclear antibodies: to penetrate    or not to penetrate, that was the question. Lupus 10, 315-318-   2. Zack, D. J., Stempniak, M., Wong, A. L., Taylor, C., and    Weisbart, R. H. (1996) Mechanisms of cellular penetration and    nuclear localization of an anti-double strand DNA autoantibody. J.    Immunol. 157, 2082-2088-   3. Hansen, J. E., Fischer, L. K., Chan, G., Chang, S. S.,    Baldwin, S. W., Aragon, R. J., Carter, J. J., Lilly, M.,    Nishimura, R. N., Weisbart, R. H., and Reeves, M. E. (2007)    Antibody-mediated p53 protein therapy prevents liver metastasis in    vivo. Cancer Res. 67, 1769-1774-   4. Hansen, J. E., Sohn, W., Kim, C., Chang, S. S., Huang, N. C.,    Santos, D. G., Chan, G., Weisbart, R. H., and    Nishimura, R. N. (2006) Antibody-mediated Hsp70 protein therapy.    Brain Res. 1088, 187-196-   5. Weisbart, R. H., Hansen, J. E., Chan, G., Wakelin, R., Chang, S.    S., Heinze, E., Miller, C. W., Koeffler, P. H., Yang, F., Cole, G.    M., Min, Y. S., and Nishimura, R. N. (2004) Antibody-mediated    transduction of p53 selectively kills cancer cells. Int. J. Oncol.    25, 1867-1873-   6. Weisbart, R. H., Hansen, J. E., Nishimura, R. N., Chan, G.,    Wakelin, R., Chang, S. S., Baresi, L., and Chamberlain, J. S. (2005)    An intracellular delivery vehicle for protein transduction of    micro-dystrophin. J. Drug Target. 13, 81-87-   7. Hansen, J. E., Chan, G., Liu, Y., Hegan, D. C., Dalal, S., Dray,    E., Kwon, Y., Xu, Y., Xu, X., Peterson-Roth, E., Geiger, E., Gera,    J., Sweasy, J. B., Sung, P., Rockwell, S., Nishimura, R. N.,    Weisbart, R. H., and P, M. G. (2012) Targeting cancer with a lupus    autoantibody. Science translational medicine 4, 157ra142-   8. Hansen, J. E., Tse, C. M., Chan, G., Heinze, E. R., Nishimura, R.    N., and Weisbart, R. H. (2007) Intranuclear protein transduction    through a nucleoside salvage pathway. J. Biol. Chem. 282,    20790-20793-   9. Zhan, X., Ander, B. P., Liao, I. H., Hansen, J. E., Kim, C.,    Clements, D., Weisbart, R. H., Nishimura, R. N., and    Sharp, F. R. (2010) Recombinant Fv-Hsp70 protein mediates    neuroprotection after focal cerebral ischemia in rats. Stroke 41,    538-543-   10. Weisbart, R. H., Wakelin, R., Chan, G., Miller, C. W., and    Koeffler, P. H. (2004) Construction and expression of a bispecific    single-chain antibody that penetrates mutant p53 colon cancer cells    and binds p53. Int. J. Oncol. 25, 1113-1118-   11. Yang, X. R., Charette, L. A., Garcia-Closas, M., Lissowska, J.,    Paal, E., Sidawy, M., Hewitt, S. M., Rimm, D. L., and    Sherman, M. E. (2006) Construction and validation of tissue    microarrays of ductal carcinoma in situ and terminal duct lobular    units associated with invasive breast carcinoma. Diagn. Mol. Pathol.    15, 157-161-   12. Chen, F. M., Epstein, A. L., Li, Z., and Taylor, C. R. (1990) A    comparative autoradiographic study demonstrating differential    intratumor localization of monoclonal antibodies to cell surface    (Lym-1) and intracellular (TNT-1) antigens. J. Nucl. Med. 31,    1059-1066-   13. Weisbart, R. H., Gera, J. F., Chan, G., Hansen, J. E., Li, E.,    Cloninger, C., Levine, A. J., and Nishimura, R. N. (2012) A    cell-penetrating bispecific antibody for therapeutic regulation of    intracellular targets. Molecular cancer therapeutics 11, 2169-2173-   14. Okudaira, K., Yoshizawa, H., and Williams, R. C., Jr. (1987)    Monoclonal murine anti-DNA antibody interacts with living    mononuclear cells. Arthritis Rheum. 30, 669-678

1. A method for selectively targeting live cells at a site of interestwith a cell-penetrating polypeptide comprises: (a) introducing orproducing extracellular DNA and its degradation product(s) near oraround the live cells at the site of interest; (b) introducing thecell-penetrating polypeptide comprising cell-penetrating determinantsnear or around the live cells, before, after or concurrently with theDNA of step (a); (c) contacting the extracellular DNA or its degradationproduct with the cell-penetrating polypeptide so that thecell-penetrating polypeptide binds the extracellular DNA or itsdegradation product near or around the live cells so as to form acomplex; (d) contacting one of the live cells with the complex in (c) soas to bind and penetrate the live cell; and (e) permitting additionalcomplexes to form as in (c) and contacting additional cells with saidcomplexes so as to bind and penetrate additional live cells at the siteof interest; thereby, selectively targeting live cells at the site ofinterest with a cell-penetrating polypeptide.
 2. (canceled)
 3. A methodfor inhibiting cellular injury in a subject comprising: (a)administering directly to the live cells at or near a site of cellularinjury of the subject a cell-penetrating polypeptide comprisingcell-penetrating determinants joined to a therapeutic agent; (b)contacting extracellular DNA or its degradation product with thecell-penetrating polypeptide so that the cell-penetrating polypeptidebinds extracellular DNA or its degradation product near or around thelive cells so as to form a complex; (c) contacting one of the live cellswith the complex in (b) so as to bind and penetrate the live cell; and(d) permitting additional complexes to form as in (b) and contactingadditional cells with said complexes so as to bind and penetrateadditional live cells at the site of cellular injury; thereby,inhibiting cellular injury in the subject.
 4. (canceled)
 5. The methodof claim 3 further comprising administering DNA to the site in anextracellular space to facilitate selective targeting at the site. 6.(canceled)
 7. A method for inhibiting a tumor associated with ischemia,cellular/tissue necrosis or cellular/tissue apoptosis by selectivetargeting of live cells at a site of interest by the method of claim 1.8.-12. (canceled)
 13. The method of claim 1, wherein thecell-penetrating polypeptide is an anti-DNA antibody.
 14. The method ofclaim 13, wherein the anti-DNA antibody binds double-stranded DNA. 15.The method of claim 13, wherein the anti-DNA antibody is selected fromthe group consisting of 3E10 antibody, 5C5 antibody, 5C6 antibody, 4H2antibody, H7 antibody, H9 antibody, H72 antibody, H205 antibody, H317antibody F14-6 antibody, SN22 antibody, SN50 antibody, SN111 antibody,SN112 antibody, SN575 antibody, SN604 antibody, SN608 antibody, F4.1antibody, J20.8 antibody, F14.6 antibody, and 9D7 antibody or a fragmentor variant thereof.
 16. The method of claim 15, wherein the anti-DNAantibody is a 3E10 antibody or a fragment or variant thereof.
 17. Themethod of claim 16, wherein the 3E10 antibody has a binding specificityof an antibody as produced by a hybridoma having ATCC accession numberPTA 2439 or a fragment or variant thereof. 18.-26. (canceled)
 27. Themethod of claim 15, wherein cell penetration is dependent on a salvagepathway. 28.-34. (canceled)
 35. The method of claim 1, wherein the cellsor tissue are as present in a mammal or derived from a mammal. 36.-42.(canceled)
 43. The method of claim 1, wherein the cell-penetratingpolypeptide is or comprises a therapeutic agent.
 44. The method of claim1, wherein the site of interest is an injury site.
 45. The method ofclaim 44, wherein the site of interest where the extracellular DNA is sointroduced is directly in the injury site.
 46. (canceled)
 47. The methodof claim 3, wherein the injury site is an intracranial injury, braininjury, myocardial infarction, skin injury, liver injury,gastrointestinal injury, lung injury, eye injury, kidney injury,pancreas injury, peritoneal injury, bone injury, nasopharyngeal injury,uterine injury, cervical injury, breast injury, organ injury, tissueinjury, burn or radiation injury.
 48. The method of claim 44, wherein aninjury in the injury site is an acute renal failure, acute organfailure, liver injury, bowel infarction, peripheral vascular disease,pulmonary failure or a cancer. 49.-60. (canceled)
 61. The method ofclaim 3, wherein the injury site is created through the use of acell-damaging agent. 62.-69. (canceled)
 70. The method of claim 1,wherein the cell-penetrating polypeptide is further linked or bound toan imaging agent or detectable marker. 71.-94. (canceled)
 95. Acomposition comprising (a) a cell-penetrating polypeptide whichcomprises cell-penetrating determinants, (b) extracellular DNA orartificial DNA and (c) a pharmaceutically acceptable carrier.
 96. Thecomposition of claim 95, wherein (a) and (b) are the sole therapeuticagents in the composition.